In summary, experimental data suggest that the concept of respiration associated with growth, mainte...

In summary, experimental data suggest that the concept of respiration associated with growth, maintenance, and ion uptake is a valuable tool in understanding the carbon balance of plants[r]

Thijs L Pons

Plant Physiological Ecology

Second Edition

Hans Lambers

The University of Western Australia Crawley, WA

Australia

hans.lambers@uwa.edu.au

F Stuart Chapin III University of Alaska Fairbanks, AK USA

terry.chapin@uaf.edu

Thijs L Pons Utrecht University The Netherlands T.L.Pons@bio.uu.nl

ISBN: 978-0-387-78340-6 e-ISBN: 978-0-387-78341-3 DOI: 10.1007/978-0-387-78341-3

Library of Congress Control Number: 2008931587

#2008 Springer ScienceỵBusiness Media, LLC

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Foreword to Second Edition

In the decade that has passed since the first edition of this book, the global environ-ment has changed rapidly Even the most steadfast ‘‘deny-ers’’ have come to accept that atmospheric CO2enrichment and global warming pose serious challenges to life on Earth Regrettably, this acceptance has been forced by calamitous events rather than by the long-standing, sober warnings of the scientific community

There seems to be growing belief that ‘‘technology’’ will save us from the worst consequences of a warmer planet and its wayward weather This hope, that may in the end prove to be no more than wishful thinking, relates principally to the built environment and human affairs Alternative sources of energy, utilized with greater efficiency, are at the heart of such hopes; even alternative ways of producing food or obtaining water may be possible For plants, however, there is no alternative but to utilize sunlight and fix carbon and to draw water from the soil (Under a given range of environmental conditions, these processes are already remarkably efficient by industrial standards.) Can we ‘‘technologize’’ our way out of the problems that plants may encounter in capricious, stormier, hotter, drier, or more saline environ-ments? Climate change will not alter the basic nature of the stresses that plants must endure, but it will result in their occurrence in places where formerly their impact was small, thus exposing species and vegetation types to more intense episodes of stress than they are able to handle The timescale on which the climate is changing is too fast to wait for evolution to come up with solutions to the problems

For a variety of reasons, the prospects for managing change seem better in agriculture than in forests or in wild plant communities It is possible to intervene dramatically in the normal process of evolutionary change by genetic manipulation Extensive screening of random mutations in a target species such as Arabidopsis thalianacan reveal genes that allow plants to survive rather simplified stress tests This is but the first of many steps, but eventually these will have their impact, primarily on agricultural and industrial crops There is a huge research effort in this area and much optimism about what can be achieved Much of it is done with little reference to plant physiology or biochemistry and has a curiously empirical char-acter One can sense that there is impatience with plant physiology that has been too slow in defining stress tolerance, and a belief that if a gene can be found that confers tolerance, and it can be transferred to a species of interest, it is not of prime

importance to know exactly what it does to the workings of the plant Such a strategy is more directed toward outcomes than understanding, even though the technology involved is sophisticated Is there a place for physiological ecology in the new order of things? The answer is perhaps a philosophical one Progress over the centuries has depended on the gradual evolution of our understanding of fundamental truths about the universe and our world Scientific discovery has always relished its serendipitous side but had we been satisfied simply with the outcomes of trial and error we would not be where we are today

It is legitimate to ask what factors set the limits on stress tolerance of a given species To answer this one must know first how the plant ‘‘works’’; in general, most of this knowledge is to hand but is based on a relatively few model species that are usually chosen because of the ease with which they can be handled in laboratory conditions or because they are economically important As well as describing the basic physiology of plants the authors of this book set out to answer more difficult questions about the differences between species with respect to environmental variables The authors would be the first to admit that comprehensive studies of comparative physiology and biochemistry are relatively few Only in a few instances we really understand how a species, or in agriculture, a genotype, pulls off the trick of surviving or flourishing in conditions where other plants fail

Of course, the above has more than half an eye on feeding the increasing world population in the difficult times that lie ahead This has to be every thinking person’s concern There is, however, more to it than that Large ecosystems interact with climate, the one affecting the other It would be as rash, for example, to ignore the effects of climate change on forests as it would be to ignore its effects on crops There is more to the successful exploitation of a given environment than can be explained exclusively in terms of a plant’s physiology An important thrust in this book is the interaction, often crucial, between plants and beneficial, pathogenic or predatory organisms that share that environment Manipulation of these interac-tions is the perennial concern of agriculture either directly or unintentionally Changes in temperature and seasonality alter established relations between organ-isms, sometimes catastrophically when, for example, a pathogen or predator expands its area of influence into plant and animal populations that have not been exposed to it previously Understanding such interactions may not necessarily allow us to avoid the worst consequences of change but it may increase our preparedness and our chances of coming up with mitigating strategies

DAVIDT CLARKSON Oak House Cheddar, UK January 2008

About the Authors

Hans Lambers is Professor of Plant Ecology and Head of School of Plant Biology at the University of Western Australia, in Perth, Australia He did his undergraduate degree at the University of Gronin-gen, the Netherlands, followed by a PhD project on effects of hypoxia on flooding-sensitive and flood-ing-tolerant Senecio species at the same institution

From 1979 to 1982, he worked as a postdoc at The University of Western Australia, Melbourne Univer-sity, and the Australian National University in Aus-tralia, working on respiration and nitrogen metabolism After a postdoc at his Alma Mater, he became Professor of Ecophysiology at Utrecht Uni-versity, the Netherlands, in 1985, where he focused on plant respiration and the physiological basis of variation in relative growth rate among herbaceous plants In 1998, he moved to the University of Wes-tern Australia, where he focuses on mineral nutri-tion and water relanutri-tions, especially in species occurring on severely phosphorus-impoverished soils in a global biodiversity hotspot He has been editor-in-chief of the journal Plant and Soil since 1992 and features on ISI’s list of highly cited authors in the field of animal and plant sciences since 2002 He was elected Fellow of the Royal Netherlands Acad-emy of Arts and Sciences in 2003

F Stuart Chapin IIIis Professor of Ecology at the Institute of Arctic Biology, University of Alaska Fairbanks, USA He did his undergraduate degree (BA) at Swarthmore College, PA, United States, and then was a Visiting Instructor in Biology (Peace Corps) at Universidad Javeriana, Bogota, Columbia, from 1966 to 1968 After that, he worked toward his PhD, on temperature compensation in phosphate absorption along a latitudinal gradient at Stanford University, United States He started at the Univer-sity of Alaska Fairbanks in 1973, focusing on plant mineral nutrition, and was Professor at this

institution from 1984 till 1989 In 1989, he became Professor of Integrative Biology, University of Cali-fornia, Berkeley, USA He returned to Alaska in 1996 His current main research focus is on effects of global change on vegetation, especially in arctic environments He features on ISI’s list of highly cited authors in ecology/environment, and was elected Member of the National Academy of Sciences, USA in 2004

Thijs L Ponsrecently retired as Senior Lecturer in Plant Ecophysiology at the Institute of Environ-mental Biology, Utrecht University, the Nether-lands He did his undergraduate degree at Utrecht University, the Netherlands, where he also worked toward his PhD, on a project on shade-tolerant and shade-avoiding species He worked in Bogor, Indo-nesia, from 1976 to 1979, on the biology of weeds in

rice Back at Utrecht University, he worked on the ecophysiology of seed dormancy and germination From the late 1980s onward he focused on photo-synthetic acclimation, including environmental sig-naling in canopies He spent a sabbatical at the University of California, Davis, USA, working with Bob Pearcy on effects of sunflecks His interest in photosynthetic acclimation was expanded to tro-pical rainforest canopies when he became involved in a project on the scientific basis of sustainable forest management in Guyana, from 1992 to 2000 He is associate editor for the journal Plant Ecology

Foreword to First Edition

The individual is engaged in a struggle for existence (Darwin) That struggle may be of two kinds: The acquisition of the resources needed for establishment and growth from a sometimes hostile and meager environment and the struggle with competing neighbors of the same or different species In some ways, we can define physiology and ecology in terms of these two kinds of struggles Plant ecology, or plant sociol-ogy, is centered on the relationships and interactions of species within communities and the way in which populations of a species are adapted to a characteristic range of environments Plant physiology is mostly concerned with the individual and its struggle with its environment At the outset of this book, the authors give their definition of ecophysiology, arriving at the conclusion that it is a point of view about physiology A point of view that is informed, perhaps, by knowledge of the real world outside the laboratory window A world in which, shall we say, the light intensity is much greater than the 200–500 mmol photons m 2s 1used in too many environment chambers, and one in which a constant 208C day and night is a great rarity The standard conditions used in the laboratory are usually regarded as treatments Of course, there is nothing wrong with this in principle; one always needs a baseline when making comparisons The idea, however, that the laboratory control is the norm is false and can lead to misunderstanding and poor predictions of behavior

The environment from which many plants must acquire resources is undergoing change and degradation, largely as a result of human activities and the relentless increase in population This has thrown the spotlight onto the way in which these changes may feed back on human well-being Politicians and the general public ask searching questions of biologists, agriculturalists, and foresters concerning the future of our food supplies, building materials, and recreational amenities The questions take on the general form, ‘‘Can you predict how ‘X’ will change when environmental variables ‘Y’ and ‘Z’ change?’’ The recent experience of experimen-tation, done at high public expense, on CO2enrichment and global warming, is a sobering reminder that not enough is known about the underlying physiology and biochemistry of plant growth and metabolism to make the confident predictions that the customers want to hear Even at the level of individual plants, there seems to be no clear prediction, beyond that the response depends on species and other ill-defined circumstances On the broader scale, predictions about the response of

plant communities are even harder to make In the public mind, at least, this is a failure The only way forward is to increase our understanding of plant metabolism, of the mechanisms of resource capture, and the way in which the captured resources are allocated to growth or storage in the plant To this extent, I can see no distinction between plant physiology and ecophysiology There are large num-bers of missing pieces of information about plant physiology—period The approach of the new millennium, then, is a good time to recognize the need to study plant physiology anew, bringing to bear the impressive new tools made available by gene cloning and recombinant DNA technology This book is to be welcomed if it will encourage ecologists to come to grips with the processes which determine the behavior of ‘‘X’’ and encourage biochemistry and physiology students to take a more realistic view of the environmental variables ‘‘Y’’ and ‘‘Z’’

The book starts, appropriately, with the capture of carbon from the atmosphere Photosynthesis is obviously the basis of life on earth, and some of the most brilliant plant scientists have made it their life’s work As a result, we know more about the molecular biophysics and biochemistry of photosynthesis than we about any other plant process The influence of virtually every environmental variable on the physiology of photosynthesis and its regulation has been studied Photosynthesis, however, occurs in an environment over which the individual plant has little control In broad terms, a plant must cope with the range of temperature, rainfall, light intensity, and CO2concentration to which its habitat is subjected It cannot change these things It must rely on its flexible physiological response to mitigate the effects of the environment At a later stage in the book, the focus shifts below ground, where the plant has rather more control over its options for capturing resources It may alter the environment around its roots in order to improve the nutrient supply It may benefit from microbial assistance in mobilizing resources or enter into more formal contracts with soil fungi and nodule-forming bacteria to acquire nutrient resources that would otherwise be unavailable or beyond its reach Toward its close, the book turns to such interactions between plants and microbes and to the chemical strategies that have evolved in plants that assist them in their struggles with one another and against browsing and grazing animals The authors end, then, on a firmly ecological note, and introduce phenomena that most labora-tory physiologists have never attempted to explore These intriguing matters remind us, as if reminders were needed, of ‘‘how little we know, how much to discover’’ (Springer and Leigh)

DAVIDT CLARKSON

IACR-Long Ashton Research Station University of Bristol

April 1997

Acknowledgments

Numerous people have contributed to the text and illustrations in this book by commenting on sections and chapters, providing photographic material, making electronic files of graphs and illustrations available, or drawing numerous figures In addition to those who wrote book reviews or sent us specific comments on the first edition of Plant Physiological Ecology, we wish to thank the following colleagues, in alphabetical order, for their valuable input: Owen Atkin, Juan Barcelo, Wilhelm Barthlott, Carl Bernacchi, William Bond, Elizabeth Bray, Siegmar Breckle, Mark Brundrett, Steve Burgess, Ray Callaway, Marion Cambridge, Art Cameron, Pilar Castro-Dı´ez, David Clarkson, Stephan Clemens, Herve Cochard, Tim Colmer, Hans Cornelissen, Marjolein Cox, Michael Cramer, Doug Darnowski, Manny Delhaize, Kingsley Dixon, John Evans, Tatsuhiro Ezawa, Jaume Flexas, Brian Forde, Peter Franks, Oula Ghannoum, Alasdair Grigg, Foteini Hassiotou, Xinhua He, Martin Heil, Angela Hodge, Richard Houghton, Rick Karban, Herbert Kronzucker, John Kuo, Jon Lloyd, Jian Feng Ma, Ken Marcum, Bjorn Martin, Justin McDonald, John Milburn, Ian Max Møller, Liesje Mommer, Ulo Niinemets, Ko Noguchi, Ram Oren, Stuart Pearse, Carol Peterson, Larry Peterson, John Pickett, Corne´ Pieterse, Bartosz Płachno, Malcolm Press, Dean Price, Miquel Ribas-Carb ´o, Peter Reich, Sarah Richardson, Peter Ryser, Yuzou Sano, Rany Schnell, Ted Schuur, Tim Setter, Michael Shane, Tom Sharkey, Sally Smith, Janet Sprent, Ernst Steudle, Youshi Tazoe, Mark Tjoelker, Robert Turgeon, David Turner, Kevin Vessey, Eric Visser, Rens Voesenek, Xianzhong Wang, Jennifer Watling, Mark Westoby, Wataru Yamori, Satoshi Yano, and Wenhao Zhang

Finally HL wishes to thank Miquel and Pepi for their fabulous hospitality when he was dealing with the final stages of the revision of the text Good company, music, food, and wine in Palma de Mallorca significantly added to the final product

HANSLAMBERS F STUARTCHAPINIII THIJSL PONS

Abbreviations

a radius of a root (ag) or root plus root hairs (ae)

A rate of CO2assimilation; also total root surface

An net rate of CO2assimilation

Af foliage area

Amax light-saturated rate of net CO2assimilation at ambient Ca

As sapwood area

ABA abscisic acid

ADP adenosine diphosphate AM arbuscular mycorrhiza AMP adenosine monophosphate

APAR absorbed photosynthetically active radiation ATP adenosine triphosphate

b individual plant biomass; buffer power of the soil B stand biomass

cs concentration of the solute

C nutrient concentration in solution; also convective heat transfer

C3 photosynthetic pathway in which the first product of CO2fixation is a 3-carbon

intermediate

C4 photosynthetic pathway in which the first product of CO2fixation is a 4-carbon

intermediate

Ca Atmospheric CO2concentration

Cc CO2concentration in the chloroplast

Ci Intercellular CO2concentration

Cli initial nutrient concentration

Cmin solution concentration at which uptake is zero

C:N carbon:nitrogen ratio CAM crassulacean acid metabolism CC carbon concentration CE carbohydrate equivalent chl chlorophyll

CPF carbon dioxide production value d plant density; also leaf dimension D diffusivity of soil water

De diffusion coefficient of ion in soil

DHAP dihydroxyacetone phosphate DM dry mass

DNA deoxyribonucleic acid

e water vapor pressure in the leaf (ei; or elin Sect 2.5 of the Chapter 4A)

or atmosphere (ea); also emissivity of a surface

E transpiration rate f tortuosity

F rate of nutrient supply to the root surface; also chlorophyll fluorescence, minimal fluorescence (F0), maximum (Fm), in a pulse of saturating light (Fm’), variable (Fv)

FAD(H2) flavine adenine dinonucleotide (reduced form)

FM fresh mass FR far-red

g diffusive conductance for CO2(gc) and water vapor (gw); boundary layer

conductance (ga); mesophyll conductance (gm); stomatal conductance (gs);

boundary layer conductance for heat transport (gah)

GA gibberellic acid GE glucose equivalent

GOGAT glutamine 2-oxoglutarate aminotransferase HCH hydroxycyclohexenone

HIR high-irradiance response

I irradiance, above (Io) or beneath (I) a canopy; irradiance absorbed; also nutrient

inflow

Imax maximum rate of nutrient inflow

IAA indoleacetic acid

IRs short-wave infrared radiation

J rate of photosynthetic electron flow

Jmax maximum rate of photosynthetic electron flow measured at saturating I and Ca

Jv water flow

k rate of root elongation; extinction coefficient for light K carrying capacity (e.g., K species)

kcat catalytic constant of an enzyme

Ki inhibitor concentration giving half-maximum inhibition

Km substrate concentration at half Vmax(or Imax)

l leaf area index

L rooting density; also latent heat of evaporation; also length of xylem element Lp root hydraulic conductance

LAI leaf area index LAR leaf area ratio LFR low-fluence response LHC light-harvesting complex LMA leaf mass per unit area LMR leaf mass ratio

LR long-wave infrared radiation that is incident (LRin), reflected (LRr), emitted

(LRem), absorbed (SRabs), or net incoming (LRnet); also leaf respiration on an area

(LRa) and mass (LRm) basis

mRNA messenger ribonucleic acid miRNA micro ribonucleic acid

M energy dissipated by metabolic processes ME malic enzyme

MRT mean residence time

Nw mol fraction, that is, the number of moles of water divided by the total number of

moles

NAD(P) nicotinamide adenine dinucleotide(phosphate) (in its oxidized form) NAD(P)H nicotinamide adenine dinucleotide(phosphate) (in its reduced form) NAR net assimilation rate

NDVI normalized difference vegetation index NEP net ecosystem production

NIR near-infrared reflectance; net rate of ion uptake NMR nuclear magnetic resonance

NPP net primary production NPQ nonphotochemical quenching

p vapor pressure

po vapor pressure of air above pure water

P atmospheric pressure; also turgor pressure Pfr far-red-absorbing configuration of phytochrome

Pi inorganic phosphate

Pr red-absorbing configuration of phytochrome

PAR photosynthetically active radiation PC phytochelatins

PEP phosphoenolpyruvate

PEPC phosphoenolpyruvate carboxylase PEPCK phosphoenolpyruvate carboxykinase

pH hydrogen ion activity; negative logarithm of the H+concentration

PGA phosphoglycerate pmf proton-motive force PNC plant nitrogen concentration PNUE photosynthetic nitrogen-use efficiency PQ photosynthetic quotient; also plastoquinone PR pathogenesis-related protein

PS photosystem

PV’ amount of product produced per gram of substrate

qN quenching of chlorophyll fluorescence due to non-photochemical processes

qP photochemical quenching of chlorophyll fluorescence

Q ubiquinone (in mitochondria), in reduced state (Qr= ubiquinol) or total quantity

(Qt); also quinone (in chloroplast)

Q10 temperature coefficient

QA primary electron acceptor in photosynthesis

r diffusive resistance, for CO2(rc), for water vapor (rw), boundary layer resistance

(ra), stomatal resistance (rs), mesophyll resistance (rm); also radial distance from

the root axis; also respiration; also growth rate (in volume) in the Lockhart equation; also proportional root elongation; also intrinsic rate of population increase (e.g., r species)

ri spacing between roots

ro root diameter

R red

R radius of a xylem element; also universal gas constant Ra molar abundance ratio of13C/12C in the atmosphere

Rd dark respiration

Rday dark respiration during photosynthesis

Re ecosystem respiration

Rp whole-plant respiration; also molar abundance ratio of13C/12C in plants

Rh heterotrophic respiration

R* minimal resource level utilised by a species RGR relative growth rate

RH relative humidity of the air RMR root mass ratio

RNA ribonucleic acid RQ respiratory quotient RR rate of root respiration RuBP ribulose-1,5-bisphosphate

Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase RWC relative water content

S nutrient uptake by roots

Sc/o specificity of carboxylation relative to oxygenation by Rubisco

SHAM salicylichydroxamic acid SLA specific leaf area SMR stem mass ratio

SR short-wave solar radiation that is incident (SRin), reflected (SRr), transmitted (SRtr),

absorbed (SRabs), used in photosynthesis (SRA), emitted in fluorescence (SRFL), or

net incoming (SRnet); also rate of stem respiration

SRL specific root length

t* time constant

tRNA transfer ribonucleic acid T temperature

TL leaf temperature

TCA tricarboxylic acid

TR total radiation that is absorbed (TRabs) or net incoming (TRnet)

u wind speed UV ultraviolet

V volume

Vc rate of carboxylation

Vo rate of oxygenation

Vcmax maximum rate of carboxylation

Vwo molar volume of water

VIS visible reflectance VLFR very low fluence response Vmax substrate-saturated enzyme activity

VPD vapor pressure deficit

w mole fraction of water vapor in the leaf (wi) or atmosphere (wa)

WUE water-use efficiency

Y yield threshold (in the Lockhart equation) g surface tension

CO2-compensation point

* CO2-compensation point in the absence of dark respiration

 boundary layer thickness; also isotopic content
isotopic discrimination

T temperature difference  elastic modulus; also emissivity  viscosity constant

 curvature of the irradiance response curve; also volumetric moisture content (mean value, ’, or at the root surface, a)

l energy required for transpiration mw chemical potential of water

mwo chemical potential of pure water under standard conditions

 Stefan–Boltzman constant

 quantum yield (of photosynthesis); also yield coefficient (in the Lockhart equation); also leakage of CO2from the bundle sheath to the mesophyll; also

relative yield of de-excitation processes water potential

air water potential of the air

m matric potential

p pressure potential; hydrostatic pressure

Contents

Foreword to Second Edition (by David T Clarkson) v

About the Authors vii

Foreword to First Edition (by David T Clarkson) ix

Acknowledgments xi

Abbreviations xiii

1 Assumptions and Approaches

Introduction7History, Assumptions, and Approaches

1 What Is Ecophysiology?

2 The Roots of Ecophysiology

3 Physiological Ecology and the Distribution of Organisms

4 Time Scale of Plant Response to Environment

5 Conceptual and Experimental Approaches

6 New Directions in Ecophysiology

7 The Structure of the Book

References

2 Photosynthesis, Respiration, and Long-Distance Transport 11

2A Photosynthesis 11

1 Introduction 11

2 General Characteristics of the Photosynthetic Apparatus 11

2.1 The ‘‘Light’’ and ‘‘Dark’’ Reactions of Photosynthesis 11

2.1.1 Absorption of Photons 12

2.1.2 Fate of the Excited Chlorophyll 13

2.1.3 Membrane-Bound Photosynthetic Electron

Transport and Bioenergetics 14

2.1.4 Photosynthetic Carbon Reduction 14

2.1.5 Oxygenation and Photorespiration 15

2.2 Supply and Demand of CO2in the Photosynthetic Process 16 2.2.1 Demand for CO27the CO27Response Curve 16 2.2.2 Supply of CO27Stomatal and Boundary Layer

Conductances 21

2.2.3 The Mesophyll Conductance 22

3 Response of Photosynthesis to Light 26

3.1 The Light Climate Under a Leaf Canopy 26

3.2 Physiological, Biochemical, and Anatomical Differences

Between Sun and Shade Leaves 27

3.2.1 The Light-Response Curve of Sun and Shade Leaves 27

3.2.2 Anatomy and Ultrastructure of Sun and Shade Leaves 29

3.2.3 Biochemical Differences Between Shade and Sun

Leaves 32

3.2.4 The Light-Response Curve of Sun and Shade

Leaves Revisited 33

3.2.5 The Regulation of Acclimation 35

3.3 Effects of Excess Irradiance 36

3.3.1 Photoinhibition7Protection by Carotenoids of the

Xanthophyll Cycle 36

3.3.2 Chloroplast Movement in Response to Changes in

Irradiance 41

3.4 Responses to Variable Irradiance 42

3.4.1 Photosynthetic Induction 43

3.4.2 Light Activation of Rubisco 43

3.4.3 Post-illumination CO2Assimilation and

Sunfleck-Utilization Efficiency 45

3.4.4 Metabolite Pools in Sun and Shade Leaves 45

3.4.5 Net Effect of Sunflecks on Carbon Gain and

Growth 47

4 Partitioning of the Products of Photosynthesis and Regulation

by ‘‘Feedback’’ 47

4.1 Partitioning Within the Cell 47

4.2 Short-Term Regulation of Photosynthetic Rate by

Feedback 48

4.3 Sugar-Induced Repression of Genes Encoding

Calvin-Cycle Enzymes 51

4.4 Ecological Impacts Mediated by Source-Sink Interactions 51

5 Responses to Availability of Water 51

5.1 Regulation of Stomatal Opening 53

5.2 The A–CcCurve as Affected by Water Stress 54 5.3 Carbon-Isotope Fractionation in Relation to Water-Use

Efficiency 56

5.4 Other Sources of Variation in Carbon-Isotope Ratios in C3

Plants 57

6 Effects of Soil Nutrient Supply on Photosynthesis 58

6.1 The Photosynthesis–Nitrogen Relationship 58

6.2 Interactions of Nitrogen, Light, and Water 59

6.3 Photosynthesis, Nitrogen, and Leaf Life Span 59

7 Photosynthesis and Leaf Temperature: Effects and Adaptations 60

7.1 Effects of High Temperatures on Photosynthesis 60

7.2 Effects of Low Temperatures on Photosynthesis 61

8 Effects of Air Pollutants on Photosynthesis 63

9 C4Plants 64

9.1 Introduction 64

9.3 Intercellular and Intracellular Transport of Metabolites

of the C4Pathway 67

9.4 Photosynthetic Efficiency and Performance at High and

Low Temperatures 68

9.5 C3–C4Intermediates 71

9.6 Evolution and Distribution of C4Species 73

9.7 Carbon-Isotope Composition of C4Species 75

10 CAM Plants 75

10.1 Introduction 75

10.2 Physiological, Biochemical, and Anatomical Aspects 76

10.3 Water-Use Efficiency 79

10.4 Incomplete and Facultative CAM Plants 79

10.5 Distribution and Habitat of CAM Species 80

10.6 Carbon-Isotope Composition of CAM Species 81

11 Specialized Mechanisms Associated with Photosynthetic

Carbon Acquisition in Aquatic Plants 82

11.1 Introduction 82

11.2 The CO2Supply in Water 82

11.3 The Use of Bicarbonate by Aquatic Macrophytes 83

11.4 The Use of CO2from the Sediment 84

11.5 Crassulacean Acid Metabolism (CAM) in Aquatic Plants 85

11.6 Carbon-Isotope Composition of Aquatic Plants 85

11.7 The Role of Aquatic Macrophytes in Carbonate

Sedimentation 85

12 Effects of the Rising CO2Concentration in the Atmosphere 87 12.1 Acclimation of Photosynthesis to Elevated CO2

Concentrations 89

12.2 Effects of Elevated CO2on Transpiration7Differential

Effects on C3, C4, and CAM Plants 90

13 Summary: What Can We Gain from Basic Principles and Rates

of Single-Leaf Photosynthesis? 90

References 91

2B Respiration 101

1 Introduction 101

2 General Characteristics of the Respiratory System 101

2.1 The Respiratory Quotient 101

2.2 Glycolysis, the Pentose Phosphate Pathway, and the

Tricarboxylic (TCA) Cycle 103

2.3 Mitochondrial Metabolism 103

2.3.1 The Complexes of the Electron-Transport Chain 104

2.3.2 A Cyanide-Resistant Terminal Oxidase 105

2.3.3 Substrates, Inhibitors, and Uncouplers 105

2.3.4 Respiratory Control 106

2.4 A Summary of the Major Points of Control of Plant

Respiration 107

2.5 ATP Production in Isolated Mitochondria and In Vivo 107

2.5.1 Oxidative Phosphorylation: The Chemiosmotic

Model 107

2.5.2 ATP Production In Vivo 107

2.6 Regulation of Electron Transport via the Cytochrome

and the Alternative Paths 109

2.6.1 Competition or Overflow? 109

2.6.2 The Intricate Regulation of the Alternative Oxidase 110

2.6.3 Mitochondrial NAD(P)H Dehydrogenases That

Are Not Linked to Proton Extrusion 112

3 The Ecophysiological Function of the Alternative Path 112

3.1 Heat Production 112

3.2 Can We Really Measure the Activity of the Alternative

Path? 113

3.3 The Alternative Path as an Energy Overflow 114

3.4 NADH Oxidation in the Presence of a High Energy Charge 117

3.5 NADH Oxidation to Oxidize Excess Redox Equivalents

from the Chloroplast 117

3.6 Continuation of Respiration When the Activity of the

Cytochrome Path Is Restricted 118

3.7 A Summary of the Various Ecophysiological Roles of the

Alternative Oxidase 118

4 Environmental Effects on Respiratory Processes 119

4.1 Flooded, Hypoxic, and Anoxic Soils 119

4.1.1 Inhibition of Aerobic Root Respiration 119

4.1.2 Fermentation 119

4.1.3 Cytosolic Acidosis 120

4.1.4 Avoiding Hypoxia: Aerenchyma Formation 121

4.2 Salinity and Water Stress 122

4.3 Nutrient Supply 123

4.4 Irradiance 123

4.5 Temperature 127

4.6 Low pH and High Aluminum Concentrations 129

4.7 Partial Pressures of CO2 130

4.8 Effects of Plant Pathogens 131

4.9 Leaf Dark Respiration as Affected by Photosynthesis 132

5 The Role of Respiration in Plant Carbon Balance 132

5.1 Carbon Balance 132

5.1.1 Root Respiration 132

5.1.2 Respiration of Other Plant Parts 133

5.2 Respiration Associated with Growth, Maintenance,

and Ion Uptake 134

5.2.1 Maintenance Respiration 134

5.2.2 Growth Respiration 136

5.2.3 Respiration Associated with Ion Transport 140

5.2.4 Experimental Evidence 140

6 Plant Respiration: Why Should It Concern Us from an

Ecological Point of View? 143

References 144

2C Long-Distance Transport of Assimilates 151

1 Introduction 151

2 Major Transport Compounds in the Phloem: Why Not Glucose? 151

3 Phloem Structure and Function 153

3.1 Symplastic and Apoplastic Transport 154

3.2 Minor Vein Anatomy 154

3.3 Sugar Transport against a Concentration Gradient 155

4 Evolution and Ecology of Phloem Loading Mechanisms 157

5 Phloem Unloading 157

6 The Transport Problems of Climbing Plants 160

7 Phloem Transport: Where to Move from Here? 161

3 Plant Water Relations 163

1 Introduction 163

1.1 The Role of Water in Plant Functioning 163

1.2 Transpiration as an Inevitable Consequence of Photosynthesis 164

2 Water Potential 165

3 Water Availability in Soil 165

3.1 The Field Capacity of Different Soils 169

3.2 Water Movement Toward the Roots 170

3.3 Rooting Profiles as Dependent on Soil Moisture Content 171

3.4 Roots Sense Moisture Gradients and Grow Toward Moist

Patches 173

4 Water Relations of Cells 174

4.1 Osmotic Adjustment 175

4.2 Cell-Wall Elasticity 175

4.3 Osmotic and Elastic Adjustment as Alternative Strategies 177

4.4 Evolutionary Aspects 178

5 Water Movement Through Plants 178

5.1 The Soil–Plant–Air Continuum 178

5.2 Water in Roots 179

5.3 Water in Stems 183

5.3.1 Can We Measure Negative Xylem Pressures? 185

5.3.2 The Flow of Water in the Xylem 186

5.3.3 Cavitation or Embolism: The Breakage of the Xylem

Water Column 188

5.3.4 Can Embolized Conduits Resume Their Function? 191

5.3.5 Trade-off Between Conductance and Safety 192

5.3.6 Transport Capacity of the Xylem and Leaf Area 194

5.3.7 Storage of Water in Stems 195

5.4 Water in Leaves and Water Loss from Leaves 196

5.4.1 Effects of Soil Drying on Leaf Conductance 196

5.4.2 The Control of Stomatal Movements and Stomatal

Conductance 199

5.4.3 Effects of Vapor Pressure Difference or Transpiration Rate

on Stomatal Conductance 201

5.4.4 Effects of Irradiance and CO2on Stomatal Conductance 203 5.4.5 The Cuticular Conductance and the Boundary Layer

Conductance 203

5.4.6 Stomatal Control: A Compromise Between Carbon Gain

and Water Loss 204

6 Water-Use Efficiency 206

6.1 Water-Use Efficiency and Carbon-Isotope Discrimination 206

6.2 Leaf Traits That Affect Leaf Temperature and Leaf Water Loss 207

6.3 Water Storage in Leaves 209

7 Water Availability and Growth 210

8 Adaptations to Drought 211

8.1 Desiccation Avoidance: Annuals and Drought-Deciduous

Species 211

8.2 Dessication Tolerance: Evergreen Shrubs 212

8.3 Resurrection Plants 212

9 Winter Water Relations and Freezing Tolerance 214

10 Salt Tolerance 216

11 Final Remarks: The Message That Transpires 216

References 217

4 Leaf Energy Budgets: Effects of Radiation and Temperature 225

4A The Plant’s Energy Balance

1 Introduction 225

2 Energy Inputs and Outputs 225

2.1 Short Overview of a Leaf’s Energy Balance 225

2.2 Short-Wave Solar Radiation 226

2.3 Long-Wave Terrestrial Radiation 229

2.4 Convective Heat Transfer 230

2.5 Evaporative Energy Exchange 232

2.6 Metabolic Heat Generation 234

3 Modeling the Effect of Components of the Energy

Balance on Leaf Temperature 234

4 A Summary of Hot and Cool Topics 235

References 235

4B Effects of Radiation and Temperature

1 Introduction 237

2 Radiation 237

2.1 Effects of Excess Irradiance 237

2.2 Effects of Ultraviolet Radiation 237

2.2.1 Damage by UV 238

2.2.2 Protection Against UV: Repair or Prevention 238

3 Effects of Extreme Temperatures 239

3.1 How Do Plants Avoid Damage by Free Radicals

at Low Temperature? 239

3.2 Heat-Shock Proteins 241

3.3 Are Isoprene and Monoterpene Emissions an Adaptation

to High Temperatures? 241

3.4 Chilling Injury and Chilling Tolerance 242

3.5 Carbohydrates and Proteins Conferring Frost

Tolerance 243

4 Global Change and Future Crops 244

References 244

5 Scaling-Up Gas Exchange and Energy Balance

from the Leaf to the Canopy Level 247

1 Introduction 247

2 Canopy Water Use 247

3 Canopy CO2Fluxes 251

4 Canopy Water-Use Efficiency 252

5 Canopy Effects on Microclimate: A Case Study 253

6 Aiming for a Higher Level 253

References 253

6 Mineral Nutrition 255

1 Introduction 255

2 Acquisition of Nutrients 255

2.1 Nutrients in the Soil 255

2.1.2 Nutrient Supply Rate 257

2.1.3 Nutrient Movement to the Root Surface 259

2.2 Root Traits That Determine Nutrient Acquisition 262

2.2.1 Increasing the Roots’ Absorptive Surface 262

2.2.2 Transport Proteins: Ion Channels and Carriers 263

2.2.3 Acclimation and Adaptation of Uptake Kinetics 265

2.2.4 Acquisition of Nitrogen 269

2.2.5 Acquisition of Phosphorus 270

2.2.6 Changing the Chemistry in the Rhizosphere 275

2.2.7 Rhizosphere Mineralization 279

2.2.8 Root Proliferation in Nutrient-Rich Patches: Is It

Adaptive? 280

2.3 Sensitivity Analysis of Parameters Involved in Phosphate

Acquisition 282

3 Nutrient Acquisition from ‘‘Toxic’’ or ‘‘Extreme’’ Soils 284

3.1 Acid Soils 284

3.1.1 Aluminum Toxicity 284

3.1.2 Alleviation of the Toxicity Symptoms by Soil

Amendment 287

3.1.3 Aluminum Resistance 287

3.2 Calcareous Soils 288

3.3 Soils with High Levels of Heavy Metals 289

3.3.1 Why Are the Concentrations of Heavy

Metals in Soil High? 289

3.3.2 Using Plants to Clean or Extract Polluted

Water and Soil: Phytoremediation and Phytomining 290

3.3.3 Why Are Heavy Metals So Toxic to Plants? 291

3.3.4 Heavy-Metal-Resistant Plants 291

3.3.5 Biomass Production of Sensitive

and Resistant Plants 296

3.4 Saline Soils: An Ever-Increasing Problem in Agriculture 296

3.4.1 Glycophytes and Halophytes 297

3.4.2 Energy-Dependent Salt Exclusion from Roots 297

3.4.3 Energy-Dependent Salt Exclusion from the Xylem 298

3.4.4 Transport of Naỵfrom the Leaves to the Roots

and Excretion via Salt Glands 298

3.4.5 Compartmentation of Salt Within the Cell

and Accumulation of Compatible Solutes 301

3.5 Flooded Soils 301

4 Plant Nutrient-Use Efficiency 302

4.1 Variation in Nutrient Concentration 302

4.1.1 Tissue Nutrient Concentration 302

4.1.2 Tissue Nutrient Requirement 303

4.2 Nutrient Productivity and Mean Residence Time 304

4.2.1 Nutrient Productivity 304

4.2.2 The Mean Residence Time of Nutrients

in the Plant 304

4.3 Nutrient Loss from Plants 306

4.3.1 Leaching Loss 306

4.3.2 Nutrient Loss by Senescence 307

4.4 Ecosystem Nutrient-Use Efficiency 308

5 Mineral Nutrition: A Vast Array of Adaptations and Acclimations 310

References 310

7 Growth and Allocation 321

1 Introduction: What Is Growth? 321

2 Growth of Whole Plants and Individual Organs 321

2.1 Growth of Whole Plants 322

2.1.1 A High Leaf Area Ratio Enables Plants to Grow Fast 322

2.1.2 Plants with High Nutrient Concentrations Can Grow

Faster 322

2.2 Growth of Cells 323

2.2.1 Cell Division and Cell Expansion: The Lockhart Equation 323

2.2.2 Cell-Wall Acidification and Removal of Calcium Reduce

Cell-Wall Rigidity 324

2.2.3 Cell Expansion in Meristems Is Controlled by Cell-Wall

Extensibility and Not by Turgor 327

2.2.4 The Physical and Biochemical Basis of Yield Threshold

and Cell-Wall Yield Coefficient 328

2.2.5 The Importance of Meristem Size 328

3 The Physiological Basis of Variation in RGR7Plants Grown with Free

Access to Nutrients 328

3.1 SLA Is a Major Factor Associated with Variation in RGR 330

3.2 Leaf Thickness and Leaf Mass Density 332

3.3 Anatomical and Chemical Differences Associated with Leaf

Mass Density 332

3.4 Net Assimilation Rate, Photosynthesis, and Respiration 333

3.5 RGR and the Rate of Leaf Elongation and Leaf Appearance 333

3.6 RGR and Activities per Unit Mass 334

3.7 RGR and Suites of Plant Traits 334

4 Allocation to Storage 335

4.1 The Concept of Storage 336

4.2 Chemical Forms of Stores 337

4.3 Storage and Remobilization in Annuals 337

4.4 The Storage Strategy of Biennials 338

4.5 Storage in Perennials 338

4.6 Costs of Growth and Storage: Optimization 340

5 Environmental Influences 340

5.1 Growth as Affected by Irradiance 341

5.1.1 Growth in Shade 341

5.1.2 Effects of the Photoperiod 345

5.2 Growth as Affected by Temperature 346

5.2.1 Effects of Low Temperature on Root Functioning 346

5.2.2 Changes in the Allocation Pattern 346

5.3 Growth as Affected by Soil Water Potential and Salinity 347

5.3.1 Do Roots Sense Dry Soil and Then Send Signals

to the Leaves? 348

5.3.2 ABA and Leaf Cell-Wall Stiffening 348

5.3.3 Effects on Root Elongation 348

5.3.4 A Hypothetical Model That Accounts for Effects

of Water Stress on Biomass Allocation 349

5.4 Growth at a Limiting Nutrient Supply 349

5.4.1 Cycling of Nitrogen Between Roots and Leaves 349

5.4.2 Hormonal Signals That Travel via the Xylem

to the Leaves 350

5.4.3 Signals That Travel from the Leaves to the Roots 351

5.4.5 Effects of Nitrogen Supply on Leaf Anatomy and

Chemistry 352

5.4.6 Nitrogen Allocation to Different Leaves, as Dependent

on Incident Irradiance 352

5.5 Plant Growth as Affected by Soil Compaction 354

5.5.1 Effects on Biomass Allocation: Is ABA Involved? 354

5.5.2 Changes in Root Length and Diameter: A Modification

of the Lockhart Equation 354

5.6 Growth as Affected by Soil Flooding 355

5.6.1 The Pivotal Role of Ethylene 356

5.6.2 Effects on Water Uptake and Leaf Growth 357

5.6.3 Effects on Adventitious Root Formation 358

5.6.4 Effects on Radial Oxygen Loss 358

5.7 Growth as Affected by Submergence 358

5.7.1 Gas Exchange 359

5.7.2 Perception of Submergence and Regulation of Shoot

Elongation 359

5.8 Growth as Affected by Touch and Wind 360

5.9 Growth as Affected by Elevated Concentrations of CO2

in the Atmosphere 361

6 Adaptations Associated with Inherent Variation in Growth Rate 362

6.1 Fast- and Slow-Growing Species 362

6.2 Growth of Inherently Fast- and Slow-Growing Species Under

Resource-Limited Conditions 363

6.2.1 Growth at a Limiting Nutrient Supply 364

6.2.2 Growth in the Shade 364

6.3 Are There Ecological Advantages Associated with a High or

Low RGR? 364

6.3.1 Various Hypotheses 364

6.3.2 Selection on RGRmaxItself, or on Traits That Are

Associated with RGRmax? 365

6.3.3 An Appraisal of Plant Distribution Requires Information

on Ecophysiology 366

7 Growth and Allocation: The Messages About Plant Messages 367

References 367

8 Life Cycles: Environmental Influences and Adaptations 375

1 Introduction 375

2 Seed Dormancy and Germination 375

2.1 Hard Seed Coats 376

2.2 Germination Inhibitors in the Seed 377

2.3 Effects of Nitrate 378

2.4 Other External Chemical Signals 378

2.5 Effects of Light 380

2.6 Effects of Temperature 382

2.7 Physiological Aspects of Dormancy 384

2.8 Summary of Ecological Aspects of Seed Germination

and Dormancy 385

3 Developmental Phases 385

3.1 Seedling Phase 385

3.2 Juvenile Phase 386

3.2.1 Delayed Flowering in Biennials 387

3.2.2 Juvenile and Adult Traits 388

3.2.3 Vegetative Reproduction 388

3.2.4 Delayed Greening During Leaf Development

in Tropical Trees 390

3.3 Reproductive Phase 391

3.3.1 Timing by Sensing Daylength: Long-Day

and Short-Day Plants 391

3.3.2 Do Plants Sense the Difference Between a Certain

Daylength in Spring and Autumn? 393

3.3.3 Timing by Sensing Temperature: Vernalization 393

3.3.4 Effects of Temperature on Plant Development 394

3.3.5 Attracting Pollinators 394

3.3.6 The Cost of Flowering 395

3.4 Fruiting 396

3.5 Senescence 397

4 Seed Dispersal 397

4.1 Dispersal Mechanisms 397

4.2 Life-History Correlates 398

5 The Message to Disperse: Perception, Transduction,

and Response 398

References 398

9 Biotic Influences 403

9A Symbiotic Associations 403

1 Introduction 403

2 Mycorrhizas 403

2.1 Mycorrhizal Structures: Are They Beneficial for Plant

Growth? 404

2.1.1 The Infection Process 408

2.1.2 Mycorrhizal Responsiveness 410

2.2 Nonmycorrhizal Species and Their Interactions

with Mycorrhizal Species 412

2.3 Phosphate Relations 413

2.3.1 Mechanisms That Account for Enhanced

Phosphate Absorption by Mycorrhizal Plants 413

2.3.2 Suppression of Colonization at High Phosphate

Availability 415

2.4 Effects on Nitrogen Nutrition 416

2.5 Effects on the Acquisition of Water 417

2.6 Carbon Costs of the Mycorrhizal Symbiosis 418

2.7 Agricultural and Ecological Perspectives 419

3 Associations with Nitrogen-Fixing Organisms 421

3.1 Symbiotic N2Fixation Is Restricted to a Fairly Limited

Number of Plant Species 422

3.2 Host–Guest Specificity in the Legume–Rhizobium

Symbiosis 424

3.3 The Infection Process in the Legume–Rhizobium

Association 424

3.3.1 The Role of Flavonoids 425

3.3.2 Rhizobial nod Genes 425

3.3.3 Entry of the Bacteria 427

3.3.4 Final Stages of the Establishment of the Symbiosis 428

3.5 Carbon and Energy Metabolism of the Nodules 431

3.6 Quantification of N2Fixation In Situ 432

3.7 Ecological Aspects of the Nonsymbiotic Association with

N2-Fixing Microorganisms 433

3.8 Carbon Costs of the Legume7Rhizobium Symbiosis 434

3.9 Suppression of the Legume7Rhizobium Symbiosis at Low pH and in the Presence of a Large Supply of

Combined Nitrogen 435

4 Endosymbionts 436

5 Plant Life Among Microsymbionts 437

References 437

9B Ecological Biochemistry: Allelopathy and Defence

against Herbivores 445

1 Introduction 445

2 Allelopathy (Interference Competition) 445

3 Chemical Defense Mechanisms 448

3.1 Defense Against Herbivores 448

3.2 Qualitative and Quantitative Defense Compounds 451

3.3 The Arms Race of Plants and Herbivores 451

3.4 How Do Plants Avoid Being Killed by Their Own Poisons? 455

3.5 Secondary Metabolites for Medicines and Crop Protection 457

4 Environmental Effects on the Production of Secondary Plant

Metabolites 460

4.1 Abiotic Factors 460

4.2 Induced Defense and Communication Between

Neighboring Plants 462

4.3 Communication Between Plants and Their Bodyguards 464

5 The Costs of Chemical Defense 466

5.1 Diversion of Resources from Primary Growth 466

5.2 Strategies of Predators 468

5.3 Mutualistic Associations with Ants and Mites 469

6 Detoxification of Xenobiotics by Plants: Phytoremediation 469

7 Secondary Chemicals and Messages That Emerge from

This Chapter 472

References 473

9C Effects of Microbial Pathogens 479

1 Introduction 479

2 Constitutive Antimicrobial Defense Compounds 479

3 The Plant’s Response to Attack by Microorganisms 481

4 Cross-Talk Between Induced Systemic Resistance and Defense

Against Herbivores 485

5 Messages from One Organism to Another 488

References 488

9D Parasitic Associations 491

1 Introduction 491

2 Growth and Development 492

2.1 Seed Germination 492

2.2 Haustoria Formation 493

2.3 Effects of the Parasite on Host Development 496

3 Water Relations and Mineral Nutrition 498

4 Carbon Relations 500

5 What Can We Extract from This Chapter? 501

References 501

9E Interactions Among Plants 505

1 Introduction 505

2 Theories of Competitive Mechanisms 509

3 How Do Plants Perceive the Presence of Neighbors? 509

4 Relationship of Plant Traits to Competitive Ability 512

4.1 Growth Rate and Tissue Turnover 512

4.2 Allocation Pattern, Growth Form, and Tissue Mass

Density 513

4.3 Plasticity 514

5 Traits Associated with Competition for Specific Resources 516

5.1 Nutrients 516

5.2 Water 517

5.3 Light 518

5.4 Carbon Dioxide 518

6 Positive Interactions Among Plants 521

6.1 Physical Benefits 521

6.2 Nutritional Benefits 521

6.3 Allelochemical Benefits 521

7 Plant7Microbial Symbiosis 522

8 Succession 524

9 What Do We Gain from This Chapter? 526

References 527

9F Carnivory 533

1 Introduction 533

2 Structures Associated with the Catching of the Prey and

Subsequent Withdrawal of Nutrients from the Prey 533

3 Some Case Studies 536

3.1 Dionaea Muscipula 537

3.2 The Suction Traps of Utricularia 539

3.3 The Tentacles of Drosera 541

3.4 Pitchers of Sarracenia 542

3.5 Passive Traps of Genlisea 542

4 The Message to Catch 543

References 543

10 Role in Ecosystem and Global Processes 545

10A Decomposition 545

1 Introduction 545

2 Litter Quality and Decomposition Rate 546

2.1 Species Effects on Litter Quality: Links with Ecological

Strategy 546

2.2 Environmental Effects on Decomposition 547

3 The Link Between Decomposition Rate and Nutrient Supply 548

3.1 The Process of Nutrient Release 548

3.2 Effects of Litter Quality on Mineralization 549

3.3 Root Exudation and Rhizosphere Effects 550

4 The End Product of Decomposition 552

10B Ecosystem and Global Processes:

Ecophysiological Controls 555

1 Introduction 555

2 Ecosystem Biomass and Production 555

2.1 Scaling from Plants to Ecosystems 555

2.2 Physiological Basis of Productivity 556

2.3 Disturbance and Succession 558

2.4 Photosynthesis and Absorbed Radiation 559

2.5 Net Carbon Balance of Ecosystems 561

2.6 The Global Carbon Cycle 561

3 Nutrient Cycling 563

3.1 Vegetation Controls over Nutrient Uptake and Loss 563

3.2 Vegetation Controls over Mineralization 565

4 Ecosystem Energy Exchange and the Hydrologic Cycle 565

4.1 Vegetation Effects on Energy Exchange 565

4.1.1 Albedo 565

4.1.2 Surface Roughness and Energy Partitioning 566

4.2 Vegetation Effects on the Hydrologic Cycle 567

4.2.1 Evapotranspiration and Runoff 567

4.2.2 Feedbacks to Climate 568

5 Moving to a Higher Level: Scaling from Physiology to the Globe 568

References 569

Glossary 573

Index 591

1

Assumptions and Approaches

Introduction—History, Assumptions, and Approaches

1 What Is Ecophysiology?

Plant ecophysiology is an experimental science that seeks to describe the physiological mechanisms underlying ecological observations In other words, ecophysiologists, or physiological ecologists, address ecological questions about the controls over the growth, reproduction, survival, abundance, and geographical distribution of plants, as these pro-cesses are affected by interactions of plants with their physical, chemical, and biotic environment These ecophysiological patterns and mechanisms can help us understand the functional significance of specific plant traits and their evolutionary heritage

The questions addressed by ecophysiologists are derived from a higher level of integration, i.e., from ‘‘ecology’’ in its broadest sense, including questions originating from agriculture, horticulture, forestry, and environmental sciences However, the ecophy-siological explanations often require mechanistic understanding at a lower level of integration (physiology, biochemistry, biophysics, molecular biology) It is, therefore, quintessential for an eco-physiologist to have an appreciation of both ecolo-gical questions and biophysical, biochemical, and molecular methods and processes In addition, many societal issues, often pertaining to agriculture, environmental change, or nature conservation, ben-efit from an ecophysiological perspective A modern ecophysiologist thus requires a good understanding of both the molecular aspects of plant processes and

the functioning of the intact plant in its environmen-tal context

2 The Roots of Ecophysiology

Plant ecophysiology aims to provide causal, mechanistic explanations for ecological questions relating to survival, distribution, abundance, and interactions of plants with other organisms Why does a particular species live where it does? How does it manage to grow there successfully, and why is it absent from other environments? These ques-tions were initially asked by geographers who described the global distributions of plants (Schimper 1898, Walter 1974) They observed consistent patterns of morphology associated with different environments and concluded that these differences in morphology must be important in explaining plant distributions Geographers, who know climatic patterns, could therefore predict the predominant life forms of plants (Holdridge 1947) For example, many desert plants have small, thick leaves that minimize the heat load and danger of overheating in hot environments, whereas shade plants often have large, thin leaves that maximize light interception These observations of morphol-ogy provided the impetus to investigate the physio-logical traits of plants from contrasting physical environments (Blackman 1919, Pearsall 1938, Ellenberg 1953, Larcher 1976)

H Lambers et al., Plant Physiological Ecology, Second edition, DOI: 10.1007/978-0-387-78341-3_1, ể Springer ScienceỵBusiness Media, LLC 2008

Although ecophysiologists initially emphasized physiological responses to the abiotic environment [e.g., to calcareous vs acidic substrates (Clarkson 1966) or dry vs flooded soils (Crawford 1978)], physiological interactions with other plants, animals, and microorganisms also benefit from an under-standing of ecophysiology As such, ecophysiology is an essential element of every ecologist’s training

A second impetus for the development of eco-physiology came from agriculture and eco-physiology Even today, agricultural production in industria-lized nations is limited to 25% of its potential by drought, infertile soils, and other environmental stresses (Boyer 1985) A major objective of agricul-tural research has always been to develop crops that are less sensitive to environmental stress so they can withstand periods of unfavorable weather or be grown in less favorable habitats For this reason agronomists and physiologists have studied the mechanisms by which plants respond to or resist environmental stresses Because some plants grow naturally in extremely infertile, dry, or salty envir-onments, ecophysiologists were curious to know the mechanisms by which this is accomplished

Plant ecophysiology is the study of physiological responses to the environment The field developed rapidly as a relatively unexplored interface between ecology and physiology Ecology provided the ques-tions, and physiology provided the tools to deter-mine the mechanism Techniques that measured the microenvironment of plants, their water relations, and their patterns of carbon exchange became typical tools of the trade in plant ecophysiology With time, these studies have explored the mechanisms of phy-siological adaptation at ever finer levels of detail, from the level of the whole plant to its biochemical and molecular bases For example, initially plant growth was described in terms of changes in plant mass Development of portable equipment for suring leaf gas exchange enabled ecologists to mea-sure rates of carbon gain and loss by individual leaves (Reich et al 1997) Growth analyses documen-ted carbon and nutrient allocation to roots and leaves and rates of production and death of individual tis-sues These processes together provide a more thor-ough explanation for differences in plant growth in different environments (Mooney 1972, Lambers & Poorter 1992) Studies of plant water relations and mineral nutrition provide additional insight into controls over rates of carbon exchange and tissue turnover More recently, we have learned many details about the biochemical basis of photosynthesis and respiration in different environments and, finally, about the molecular basis for differences in key photosynthetic and respiratory proteins This

mainstream of ecophysiology has been highly suc-cessful in explaining why plants are able to grow where they

3 Physiological Ecology

and the Distribution of Organisms

Although there are 270000 species of land plants (Hammond 1995), a series of filters eliminates most of these species from any given site and restricts the actual vegetation to a relatively small number of species (Fig 1) Many species are absent from a given plant community for historical rea-sons They may have evolved in a different region and never dispersed to the site under consideration For example, the tropical alpine of South America has few species in common with the tropical alpine of Africa, despite similar environmental conditions, whereas eastern Russia and Alaska have very simi-lar species composition because of extensive migra-tion of species across a land bridge connecting these regions when Pleistocene glaciations lowered sea level 20000—100000 years ago

Of those species that arrive at a site, many lack the appropriate physiological traits to survive the physical environment For example, whalers inad-vertently brought seeds of many weedy species to Svalbard, north of Norway, and to Barrow, in north-ern Alaska However, in contrast to most temperate regions, there are currently no exotic weed species in these northern sites (Billings 1973) Clearly, the physical environmenthas filtered out many species that may have arrived but lacked the physiological traits to grow, survive, and reproduce in the Arctic Biotic interactionsexert an additional filter that eliminates many species that may have arrived and are capable of surviving the physical environment Most plant species that are transported to different continents as ornamental or food crops never spread beyond the areas where they were planted because they cannot compete with native species (a biotic filter) Sometimes, however, a plant species that is introduced to a new place without the diseases or herbivores that restricted its distribution in its native habitat becomes an aggressive invader, for example, Opuntia ficus-indica (prickly pear) in Aus-tralia, Solidago canadensis (golden rod) in Europe, Cytisus scoparius(Scotch broom) in North America, and Acacia cyclops (red-eyed wattle) and A saligna (orange wattle) in South Africa Because of biotic interactions, the actual distribution of a species (rea-lized niche, as determined by ecological amplitude) is more restricted than the range of conditions

where it can grow and reproduce (its fundamental niche, as determined by physiological amplitude) (Fig 2)

Historical, physiological, and biotic filters are constantly changing and interacting Human and natural introductions or extinctions of species, chance dispersal events, and extreme events such as volcanic eruptions or floods change the species pool present at a site Changes in climate, weath-ering of soils, and introduction or extinction of species change the physical and biotic environ-ment Those plant species that can grow and repro-duce under the new conditions or respond evolutionarily so that their physiology provides a better match to this environment will persist Because of these interacting filters, the species pre-sent at a site are simply those that arrived and survived There is no reason to assume that the species present at a site attain their maximal phy-siologically possible rates of growth and reproduc-tion (Vrba & Gould 1986) In fact, controlled-environment studies typically demonstrate that a given species is most common under environmen-tal conditions that are distinctly suboptimal for

most physiological processes because biotic inter-actions prevent most species from occupying the most favorable habitats (Fig 2)

Given the general principle that species that are present at any site reflect their arrival and survival, why does plant species diversity differ among sites that differ in soil fertility? Typically, this diversity increases with decreasing soil fertility, up to a max-imum, and then declines again (Grime 1979, Huston 1994) To answer this question, we need detailed ecophysiological information on the various mechanisms that allow plants to compete and co-exist in different environments The information that is required will depend on which ecosystem is studied In biodiverse (i.e., species-rich), nutrient-poor, tropical rainforests, with a wide variation in light climate, plant traits that enhance the conver-sion of light into biomass or conserve carbon are likely to be important for an understanding of plant diversity In the biodiverse, nutrient-impover-ished sandplains of South Africa and Australia, however, variation in root traits that are associated with nutrient acquisition offers clues to understand-ing plant species diversity

FIGURE Historical, physiological, and

4 Time Scale of Plant Response to Environment

We define stress as an environmental factor that reduces the rate of some physiological process (e.g., growth or photosynthesis) below the maxi-mum rate that the plant could otherwise sustain Stresses can be generated by abiotic and/or biotic processes Examples of stress include low nitrogen availability, heavy metals, high salinity, and shad-ing by neighborshad-ing plants The immediate response of the plant to stress is a reduction in performance (Fig 3) Plants compensate for the detrimental effects of stress through many mechanisms that operate over different time scales, depending on the nature of the stress and the physiological processes that are affected Together, these compen-satory responses enable the plant to maintain a rela-tively constant rate of physiological processes despite occurrence of stresses that periodically reduce performance If a plant is going to be success-ful in a stresssuccess-ful environment, then there must be some degree of stress resistance Mechanisms of

stress resistance differ widely among species They range from avoidance of the stress, e.g., in deep-rooting species growing in a low-rainfall area, to stress tolerance, e.g., in Mediterranean species that can cope with a low leaf water content

Physiological processes differ in their sensitivity to stress The most meaningful physiological pro-cesses to consider are growth and reproduction, which integrate the stress effects on fine-scale phy-siological processes as they relate to fitness, i.e., differential survival and reproduction in a competi-tive environment To understand the mechanism of plant response, however, we must consider the response of individual processes at a finer scale (e.g., the response of photosynthesis or of light-har-vesting pigments to a change in light intensity) We recognize at least three distinct time scales of plant response to stress:

1 The stress response is the immediate detrimental effect of a stress on a plant process This generally occurs over a time scale of seconds to days, resulting in a decline in performance of the process

FIGURE2 Biomass production of two hypothetical spe-cies (x and y) as a function of resource supply In the absence of competition (upper panels), the physiologi-cal amplitude of species x and y (PAxand PAy,

respec-tively) defines the range of conditions over which each species can grow In the presence of competition (lower panels), plants grow over a smaller range of conditions (their ecological amplitude, EAx and EAy) that is

constrained by competition from other species A given pattern of species distribution (e.g., that shown in the bottom panels) can result from species that differ in their maximum biomass achieved (left-hand pair of graphs), shape of resource response curve (center pair of graphs), or physiological amplitude (right-hand pair of graphs) Adapted from Walter (1973)

2 Acclimation is the morphological and physiolo-gical adjustment by individual plants to compen-sate for the decline in performance following the initial stress response Acclimation occurs in response to environmental change through changes in the activity or synthesis of new bio-chemical constituents such as enzymes, often associated with the production of new tissue These biochemical changes then initiate a cascade of effects that are observed at other levels, such as changes in rate or environmental sensitivity of a specific process (e.g., photosynthesis), growth rate of whole plants, and morphology of organs or the entire plant Acclimation to stress always occurs within the lifetime of an individual, usually within days to weeks Acclimation can be demonstrated by comparing genetically simi-lar plants that are growing in different environments

3 Adaptation is the evolutionary response result-ing from genetic changes in populations that compensate for the decline in performance caused by stress The physiological mechanisms of response are often similar to those of acclima-tion, because both require changes in the activity or synthesis of biochemical constituents and cause changes in rates of individual physiologi-cal processes, growth rate, and morphology In

fact, adaptation may alter the potential of plants to acclimate to short-term environmental varia-tion Adaptation, as we define it, differs from acclimation in that it requires genetic changes in populations and therefore typically requires many generations to occur We can study adapta-tion by comparing genetically distinct plants grown in a common environment

Not all genetic differences among populations reflect adaptation Evolutionary biologists have often criticized ecophysiologists for promoting the ‘‘Panglossian paradigm’’, i.e., the idea that just because a species exhibits certain traits in a particu-lar environment, these traits must be beneficial and must have resulted from natural selection in that environment (Gould & Lewontin 1979) Plants may differ genetically because their ancestral species or populations were genetically distinct before they arrived in the habitat we are studying or other his-torical reasons may be responsible for the existence of the present genome Such differences are not necessarily adaptive

There are at least two additional processes that can cause particular traits to be associated with a given environment:

1 Through the quirks of history, the ancestral spe-cies or population that arrived at the site may

FIGURE3 Typical time course of plant response to envir-onmental stress The immediate response to environ-mental stress is a reduction in physiological activity Through acclimation, individual plants compensate for this stress such that activity returns toward the control level Over evolutionary time, populations adapt to environmental stress, resulting in a further increase in

have been pre-adapted, i.e., exhibited traits that allowed continued persistence in these condi-tions Natural selection for these traits may have occurred under very different environmental cir-cumstances For example, the tree species that currently occupy the mixed deciduous forests of Europe and North America were associated with very different species and environments during the Pleistocene, 100000 years ago They co-occur now because they migrated to the same place some time in the past (the historical filter), can grow and reproduce under current environmen-tal conditions (the physiological filter), and out-competed other potential species in these communities and successfully defended them-selves against past and present herbivores and pathogens (the biotic filter)

2 Once species arrive in a given geographic region, their distribution is fine-tuned by ecological sort-ing, in which each species tends to occupy those habitats where it most effectively competes with other plants and defends itself against natural enemies (Vrba & Gould 1986)

5 Conceptual and Experimental Approaches

Documentation of the correlation between plant traits and environmental conditions is the raw material for many ecophysiological questions Plants in the high alpine of Africa are strikingly similar in morphology and physiology to those of the alpine of tropical South America and New Guinea, despite very different phylogenetic his-tories The similarity of physiology and morphol-ogy of shrubs from Mediterranean regions of western parts of Spain, South Africa, Chile, Australia, and the United States suggests that the distinct floras of these regions have undergone convergent evolution in response to similar cli-matic regimes (Mooney & Dunn 1970) For exam-ple, evergreen shrubs are common in each of these regions These shrubs have small, thick leaves, which continue to photosynthesize under condi-tions of low water availability during the warm, dry summers characteristic of Mediterranean cli-mates The shrubs of all Mediterranean regions effectively retain nutrients when leaves are shed, a trait that could be important on infertile soils, and often resprout after fire, which occurs com-monly in these regions Documentation of a corre-lation of traits with environment, however, can

never determine the relative importance of adapta-tion to these condiadapta-tions and other factors such as pre-adaptation of the ancestral floras and ecologi-cal sorting of ancestral species into appropriate habitats Moreover, traits that are measured under field conditions reflect the combined effects of differences in magnitude and types of environ-mental stresses, genetic differences among popula-tions in stress response, and acclimation of individuals to stress Thus, documentation of cor-relations between physiology and environment in the field provides a basis for interesting ecophy-siological hypotheses, but these hypotheses can rarely be tested without complementary approaches such as growth experiments or phylo-genetic analyses

Growth experiments allow one to separate the effects of acclimation by individuals and genetic differences among populations Acclimation can be documented by measuring the physiology of genetically similar plants grown under different environmental conditions Such experiments show, for example, that plants grown at low perature generally have a lower optimum tem-perature for photosynthesis than warm-grown plants (Billings et al 1971) By growing plants col-lected from alpine and low-elevation habitats under the same environmental conditions, we can demonstrate genetic differences: with the alpine plant generally having a lower temperature opti-mum for photosynthesis than the low-elevation population Thus, many alpine plants photo-synthesize just as rapidly as their low-elevation counterparts, due to both acclimation and adapta-tion Controlled-environment experiments are an important complement to field observations Conversely, field observations and experiments provide a context for interpreting the significance of laboratory experiments

Both acclimation and adaptation reflect complex changes in many plant traits, making it difficult to evaluate the importance of changes in any particular trait Ecological modeling and molecular modifica-tion of specific traits are two approaches to explore the ecological significance of specific traits Ecologi-cal models can range from simple empiriEcologi-cal rela-tionships (e.g., the temperature response of photosynthesis) to complex mathematical models that incorporate many indirect effects, such as nega-tive feedbacks of sugar accumulation to photosynth-esis A common assumption of these models is that there are both costs and benefits associated with a particular trait, such that no trait enables a plant to perform best in all environments (i.e., there are no ‘‘super-plants’’ or ‘‘Darwinian demons’’ that are

superior in all components) That is presumably why there are so many interesting physiological differences among plants These models seek to identify the conditions under which a particular trait allows superior performance or compare per-formance of two plants that differ in traits The theme of trade-offs (i.e., the costs and benefits of particular traits) is one that will recur frequently in this book

A second, more experimental approach to the question of optimality is molecular modification of the gene that encodes a trait, including the reg-ulation of its expression In this way we can explore the consequences of a change in photosynthetic capacity, sensitivity to a specific hormone, or response to shade This molecular approach is an extension of comparative ecophysiological studies, in which plants from different environments that are as similar as possible except with respect to the trait of interest are grown in a common environment Molecular modification of single genes allows eva-luation of the physiological and ecological conse-quences of a trait, while holding constant the rest of the biology of the plants

6 New Directions in Ecophysiology

Plant ecophysiology has several new and poten-tially important contributions to make to biology The rapidly growing human population requires increasing supplies of food, fiber, and energy, at a time when the best agricultural land is already in production or being lost to urban development and land degradation It is thus increasingly critical that we identify traits or suites of traits that maximize sustainable food and fiber production on both highly productive and less productive sites The development of varieties that grow effectively with inadequate supplies of water and nutrients is parti-cularly important in less developed countries that often lack the economic and transportation resources to support high-intensity agriculture Molecular biology and traditional breeding pro-grams provide the tools to develop new combina-tions of traits in plants, including GMOs (genetically modified organisms) Ecophysiology is perhaps the field that is best suited to determine the costs, ben-efits, and consequences of changes in these traits, as whole plants, including GMOs, interact with com-plex environments

Past ecophysiological studies have described important physiological differences among plant species and have demonstrated many of the

mechanisms by which plants can live where they occur These same physiological processes, how-ever, have important effects on the environment, shading the soil, removing nutrients that might otherwise be available to other plants or soil micro-organisms, transporting water from the soil to the atmosphere, thus both drying the soil and increas-ing atmospheric moisture These plant effects can be large and provide a mechanistic basis for under-standing processes at larger scales, such as commu-nity, ecosystem, and climatic processes (Chapin 2003) For example, forests that differ only in species composition can differ substantially in productivity and rates of nutrient cycling Simulation models suggest that species differences in stomatal conduc-tance and rooting depth could significantly affect climate at regional and continental scales (Foley et al 2003, Field et al 2007) As human activities increasingly alter the species composition of large portions of the globe, it is critical that we understand the ecophysiological basis of community, ecosys-tem, and global processes

7 The Structure of the Book

the processes up to the level of an entire canopy, demonstrating that processes at the level of a canopy are not necessarily the sum of what happens in single leaves, due to the effects of the surrounding leaves (Chapter 5) Chapter discusses mineral nutrition and the numerous ways in which plants cope with soils with low nutrient availability or toxic metal concentrations (e.g., sodium, aluminum, heavy metals) These first chapters emphasize those aspects that help us to analyze ecological problems Moreover, they provide a sound basis for later chap-ters in the book that deal with a higher level of integration

The following chapters deal with patterns of growth and allocation (Chapter 7), life-history traits (Chapter 8), and interactions of individual plants with other organisms: symbiotic microorganisms (Chapter 9A); ecological biochemistry, discussing allelopathy and defense against herbivores (Chapter 9B); microbial pathogens (Chapter 9C); parasitic plants (Chapter 9D); interactions among plants in communities (Chapter 9E); and animals used as prey by carnivorous plants (Chapter 9F) These chapters build on information provided in the initial chapters

The final chapters deal with ecophysiological traits that affect decomposition of plant material in contrasting environments (Chapter 10A) and with the role of plants in ecosystem and global processes (Chapter 10B) Many topics in the first two series of chapters are again addressed in this broader context For example, allocation patterns and defense com-pounds affect decomposition Photosynthetic path-ways and allocation patterns affect to what extent plant growth is enhanced at elevated levels of car-bon dioxide in the atmosphere

Throughout the text, ‘‘boxes’’ are used to elabo-rate on specific problems, without cluttering up the text They are meant for students seeking a deeper understanding of problems discussed in the main text A glossary provides quick access to the mean-ing of technical terms used in both this book and the plant ecophysiological literature The references at the end of each chapter are an entry point to relevant literature in the field We emphasize review papers that provide broad syntheses but also include key experimental papers in rapidly developing areas (‘‘the cutting edge’’) In general, this book aims at students who are already familiar with basic princi-ples in ecology, physiology, and biochemistry It should provide an invaluable text for both under-graduates and postunder-graduates and a reference for professionals

References

Billings, W.D 1973 Arctic and alpine vegetation: Simila-rities, differences, and susceptibility to disturbance BioScience 23: 697—704

Billings, W.D., Godfrey, P.J., Chabot, B.F., & Bourque, D.P 1971 Metabolic acclimation to temperature in arctic and alpine ecotypes of Oxyria digyna Arc Alp Res 3: 277—289 Blackman, V.H 1919 The compound interest law and plant

growth Ann Bot 33: 353—360

Boyer, J.S 1985 Water transport Annu Rev Plant Physiol 36: 473—516

Chapin III, F.S., 2003 Effects of plant traits on ecosystem and regional processes: A conceptual framework for predicting the consequences of global change Ann Bot 91: 455—463 Clarkson, D.T 1966 Aluminium tolerance in species

within the genus Agrostis J Ecol 54: 167—178

Crawford, R.M.M 1978 Biochemical and ecological simi-larities in marsh plants and diving animals Naturwis-senschaften 65: 194201

Ellenberg, H 1953 Physiologisches und ăokologisches Ver-halten derselben Pflanzanarten Ber Deutsch Bot Ges 65: 351—361

Field, C.B., Lobell, D.B., Peters, H.A., & Chiariello, N.R 2007 Feedbacks of terrestrial ecosystems to climate change Annu Rev Env Res 32: 1—29

Foley, J.A., Costa, M.H., Delire, C., Ramankutty, N., & Snyder, P 2003 Green surprise? How terrestrial ecosys-tems could affect earth’s climate Front Ecol Environ 1: 38—44

Gould, S.J & Lewontin, R.C 1979 The spandrels of San Marco and the Panglossian paradigm: A critique of the adaptationists programme Proc R Soc Lond B 205: 581—598

Grime, J.P 1979 Plant strategies and vegetation processes Wiley, Chichester

Hammond, P.M 1995 The current magnitude of biodiversity In: Global biodiversity assessment, V.H Heywood (ed.) Cambridge University Press, Cambridge, pp 113—138 Holdridge, L.R 1947 Determination of world plant

forma-tions from simple climatic data Science 105: 367—368 Huston, M.A 1994 Biological diversity Cambridge

Uni-versity Press, Cambridge

Lambers, H & Poorter, H 1992 Inherent variation in growth rate between higher plants: A search for physio-logical causes and ecophysio-logical consequences Adv Ecol Res 22: 187—261

Larcher, W 1976 ¨Okologie der Pflanzen Ulmer, Stuttgart Mooney, H.A 1972 The carbon balance of plants Annu

Rev Ecol Syst 3: 315—346

Mooney, H.A & Dunn, E.L 1970 Convergent evolution of Mediterranean-climate sclerophyll shrubs Evolution 24: 292—303

Pearsall, W.H 1938 The soil complex in relation to plant communities J Ecol 26: 180—193

Reich, P.B., Walters, M.B., & Ellsworth, D.S 1997 From tropics to tundra: Global convergence in plant function-ing Proc Natl Acad Sci 94: 13730—13734

Schimper, A.F.W 1898 Pflanzengeographie und phy-siologische Grundlage Verlag von Gustav Fischer, Jena Vrba, E.S & Gould, S.J 1986 The hierarchical expansion of sorting and selection: Sorting and selection cannot be equated Paleobiology 12: 217—228

Walter, H 1973 Die Vegetation der Erde in oăkophysio-logischer Betrachtung 3rd ed Gutsav Fisher Verlag, Jena

2

Photosynthesis, Respiration, and Long-Distance Transport

2A Photosynthesis

1 Introduction

Approximately 40% of a plant’s dry mass consists of carbon, fixed in photosynthesis This process is vital for growth and survival of virtually all plants dur-ing the major part of their growth cycle In fact, life on Earth in general, not just that of plants, totally depends on current and/or past photosynthetic activity Leaves are beautifully specialized organs that enable plants to intercept light necessary for photosynthesis The light is captured by a large array of chloroplasts that are in close proximity to air and not too far away from vascular tissue, which supplies water and exports the products of photo-synthesis In most plants, CO2 uptake occurs through leaf pores, the stomata, which are able to rapidly change their aperture (Sect 5.4 of Chapter on plant water relations) Once inside the leaf, CO2 diffuses from the intercellular air spaces to the sites of carboxylation in the chloroplast (C3species) or in the cytosol (C4and CAM species)

Ideal conditions for photosynthesis include an ample supply of water and nutrients to the plant, and optimal temperature and light conditions Even when the environmental conditions are less favorable, however, such as in a desert, alpine environments, or the understory of a forest, photo-synthesis, at least of the adapted and acclimated plants, continues (for a discussion of the concepts of acclimation and adaptation, see Fig and

Sect in Chapter on assumptions and approaches) This chapter addresses how such plants manage to photosynthesize and/or protect their photosynthetic machinery in adverse envir-onments, what goes wrong in plants that are not adapted and fail to acclimate, and how photosynth-esis depends on a range of other physiological activities in the plant

2 General Characteristics of the Photosynthetic Apparatus

2.1 The ‘‘Light’’ and ‘‘Dark’’ Reactions of Photosynthesis

To orient ourselves, we imagine zooming in on a chloroplast: from a tree, to a leaf, to a cell in a leaf, and then to the many chloroplasts in a single cell, where the primary processes of photosynthesis occur In C3 plants most of the chloroplasts are located in the mesophyll cells of the leaves (Fig 1) Three main processes are distinguished:

1 Absorption of photons by pigments, mainly chlorophylls, associated with two photosystems The pigments are embedded in internal mem-brane structures (thylakoids) and absorb a major part of the energy of the photosynthetically

H Lambers et al., Plant Physiological Ecology, Second edition, DOI: 10.1007/978-0-387-78341-3_2, ể Springer ScienceỵBusiness Media, LLC 2008

active radiation (PAR; 400—700 nm) They transfer the excitation energy to the reaction centers of the photosystems where the second process starts Electrons derived from the splitting of water with

the simultaneous production of O2 are trans-ported along an electron-transport chain embedded in the thylakoid membrane NADPH and ATP produced in this process are used in the third process Since these two reactions depend on light energy, they are called the ‘‘light reac-tions’’of photosynthesis

3 The NADPH and ATP are used in the photosyn-thetic carbon-reduction cycle (Calvin cycle), in which CO2is assimilated leading to the synthesis of C3compounds (triose-phosphates) These pro-cesses can proceed in the absence of light and are referred to as the ‘‘dark reactions’’ of photosynth-esis As discussed in Sect 3.4.2, however, some of the enzymes involved in the ‘‘dark’’ reactions require light for their activation, and hence the

difference between ‘‘light’’ and ‘‘dark’’ reaction is somewhat blurred

2.1.1 Absorption of Photons

The reaction center of photosystem I (PS I) is a chlorophyll dimer with an absorption peak at 700 nm, hence called P700 There are about 110 ‘‘ordinary’’ chlorophyll a (chl a) molecules per P700 as well as several different protein molecules, to keep the chlorophyll molecules in the required posi-tion in the thylakoid membranes (Lichtenthaler & Babani 2004) The number of PS I units can be quan-tified by determining the amount of P700molecules, which can be assessed by measuring absorption changes at 830 nm

The reaction center of photosystem II (PS II) contains redox components, including a chlorophyll amolecule with an absorption peak at 680 nm, called

FIGURE (A) Scanning electron microscope

cross-sectional view of a dorsiventral leaf of Nicotiana taba-cum (tobacco), showing palisade tissue beneath the upper (adaxial) epidermis, and spongy tissue adjacent to the (lower) abaxial epidermis (B) Scanning electron microscope cross-sectional view of an isobilateral leaf of Hakea prostrata (harsh hakea) (C) Transmission elec-tron microscope micrograph of a tobacco chloroplast, showing appressed (grana) and unappressed regions of

P680, pheophytin, which is like a chlorophyll mole-cule but without the Mg atom, and the first quinone acceptor of an electron (QA) (Chow 2003) Redox cofactors in PS II are bound to the structure of the so-called D1/D2 proteins in PS II PS I and PS II units not contain chl b (Lichtenthaler & Babani 2004) Several protein molecules keep the chlorophyll molecules in the required position in the thylakoid membranes In vitro, P680is too unstable to be used to quantify the amount of PS II The herbicide atra-zine binds specifically to one of the complexing protein molecules of PS II, however; when using 14

C-labeled atrazine, this binding can be quantified and used to determine the total amount of PS II Alternatively, the quantity of functional PS II centers can be determined, in vivo, by the O2yield from leaf disks, exposed to 1% CO2 and repetitive light flashes A good correlation exists between the two assays The O2yield per flash provides a convenient, direct assay of PS II in vivo when conditions are selected to avoid limitation by PS I (Chow et al 1989)

A large part of the chlorophyll is located in the light-harvesting complex(LHC) These chlorophyll molecules act as antennae to trap light and transfer its excitation energy to the reaction centers of one of

the photosystems The reaction centers are strategi-cally located to transfer electrons along the electro-n-transport chains The ratio of chl a/chl b is about 1.1—1.3 for LHC (Lichtenthaler & Babani 2004)

Leaves appear green in white light, because chlorophyll absorbs more efficiently in the blue and red than in the green portions of the spectrum; beyond approximately 720 nm, there is no absorp-tion by chlorophyll at all The absorpabsorp-tion spectrum of intact leaves differs from that of free chlorophyll in solution, and leaves absorb a significant portion of the radiation in regions where chlorophyll absorbs very little in vitro (Fig 2) This is due to (1) the modification of the absorption spectra of the chlorophyll molecules bound in protein complexes in vivo, (2) the presence of accessory pigments, such as carotenoids, in the chloroplast, and, most impor-tantly, (3) light scattering within the leaf (Sect 3.2.2)

2.1.2 Fate of the Excited Chlorophyll

Each quantum of red light absorbed by a chloro-phyll molecule raises an electron from a ground state to an excited state Absorption of light of shorter wavelengths (e.g., blue light) excites the chlorophyll to an even higher energy state In the

FIGURE2 (A) The relative absorbance spectrum of

chlor-ophyll a and chlorchlor-ophyll b; absorbance = –log (trans-mitted light/incident light); (B) The relative absorbance spectrum of pigment-protein complexes: PS II reaction centre and PS II light-harvesting complex; (courtesy J.R Evans, Research School of Biological Sciences, Australian National University, Canberra, Australia

(C) Light absorption of an intact green leaf of Encelia californica; for comparison the absorption spectrum of an intact white (pubescent) leaf of Encelia farinosa (brittlebush) is also given From Ehleringer et al (1976), Science 227: 1479–1481 Reprinted with kind permission from AAS

higher energy state after absorption of blue light, however, chlorophyll is unstable and rapidly gives up some of its energy to the surroundings as heat, so that the elevated electron immediately falls back into the orbit of the electron excited by red light Thus, whatever the wavelength of the light absorbed, chlorophyll reaches the same excitation state upon photon capture In this excitation state, chlorophyll is stable for 10—9seconds, after which it disposes of its available energy in one of three ways (Krause & Weis 1991):

1 The primary pathway of excitation energy is its highly efficient transfer to other chlorophyll molecules, and ultimately to the reaction center where it is used in photochemistry, driving bio-chemical reactions

2 The excited chlorophyll can also return to its ground state by converting its excitation energy into heat In this process no photon is emitted The excited chlorophyll can emit a photon and

thereby return to its ground state; this process is called fluorescence Most fluorescence is emitted by chl a of PS II The wavelength of fluorescence is slightly longer than that of the absorbed light, because a portion of the excitation energy is lost before the fluorescence photon is emitted Chlorophylls usually fluoresce in the red; it is a deeper red (the wavelength is about 10 nm longer) than the red absorption peak of chloro-phyll Fluorescence increases when photochem-istry and/or dissipation are low relative to photon absorption, but the process is not regu-lated as such This can occur under conditions of excessive light, severely limiting CO2supply, or stresses that inhibit photochemistry

The primary photochemical reactions of PS II and PS I occur at a much faster rate than subsequent electron transport (Sect 2.1.3), which in turn occurs faster than carbon reduction processes (Sect 2.1.4) Since the three compartments of the photosynthetic apparatus operate in series, they are each tightly regulated to coordinate their activity under chan-ging conditions

2.1.3 Membrane-Bound Photosynthetic Electron Transport and Bioenergetics

The excitation energy captured by the pigments is transferred to the reaction centers of PS I and PS II PS I and PS II are associated with different regions of the thylakoid membrane PS I is located in the stroma-exposed ‘‘unappressed’’ regions, and PS II is largely associated with the ‘‘appressed’’ regions

where thylakoids border other thylakoids (grana) (Fig 1) In PS II an electron, derived from the split-ting of water into O2and protons, is transferred to pheophytin, and then to plastoquinone (QA, bound to D2 protein, a one-electron carrier), followed by transfer to QB(bound to D1 protein, a two-electron carrier), and then to free plastoquinone Plastoqui-none (PQ) is subsequently reduced and transported to the cytochrome b/f complex In the process pro-tons are transported across the membrane into the thylakoid lumen (Fig 3) The two sources of protons acidify and charge the thylakoid lumen positively The electrochemical potential gradient across the thylakoid membrane, representing a proton-motive force, is subsequently used to phosphorylate ADP, thus producing ATP This reaction is catalyzed by an ATPase, or coupling factor, located in the stroma-exposed, unappressed regions of the thylakoids In linear electron transport, electrons are transferred from the cytochrome b/f complex to PS I through plastocyanin (PC) that migrates through the thyla-koid lumen NADP is reduced by ferredoxin as the terminal acceptor of electrons from PS I which results in formation of NADPH In cyclic electron transport, electrons are transferred from PS I back to cytochrome b/f through plastoquinone, thus contri-buting to proton extrusion in the lumen and subse-quent ATP synthesis NADPH and ATP are used in the carbon-reduction cycle that is located in the stroma Linear electron transport is the principal pathway, whereas the engagement of cyclic electron transport is tuned to the demand for ATP relative to NADPH Other components of the photosynthetic membrane are also regulated, particularly with respect to the prevailing light conditions

2.1.4 Photosynthetic Carbon Reduction

reactions that are part of the Calvin cycle in which ATP and NADPH are consumed (Fig 4) About 1/6 of the triose-P remaining in the chloroplast is used to produce starch, which is stored inside the chloro-plast, or is exported During the night, starch may be hydrolyzed, and the product of this reaction, triose-P, is exported to the cytosol The photosynthetic carbon-reduction cycle has various control points and factors that function as stabilizing mechanisms under changing environmental conditions

2.1.5 Oxygenation and Photorespiration

Rubisco catalyzes not only the carboxylation of RuBP, but also its oxygenation (Fig 5) The ratio of the carboxylation and the oxygenation reaction strongly depends on the relative concentrations of CO2and O2and on leaf temperature The products of the carboxylation reaction are two C3 mole-cules (PGA), whereas the oxygenation reaction produces only one PGA and one C2 molecule:

phosphoglycolate This C2molecule is first depho-sphorylated in the chloroplast, producing glycolate (Fig 5), which is exported to the peroxisomes, where it is metabolized to glyoxylate and then glycine Glycine is exported to the mitochondria where two molecules are converted to produce one serine with the release of one molecule of CO2and one NH3 Serine is exported back to the peroxisomes, where a transamination occurs, producing one molecule of hydroxypyruvate and then glycerate Glycerate moves back to the chloroplast, to be converted into PGA So, out of two phosphoglycolate molecules one glycerate is made and one C-atom is lost as CO2 The entire process, starting with the oxygena-tion reacoxygena-tion, is called photorespiraoxygena-tion, as it con-sumes O2and releases CO2; it depends on light, or, more precisely, on photosynthetic activity The pro-cess is distinct from ‘‘dark respiration’’ that largely consists of mitochondrial decarboxylation processes that proceed independent of light Dark respiration is discussed in Chapter 2B on respiration

FIGURE Schematic representation of the thylakoid

membrane, enclosing the thylakoid lumen, showing the transfer of excitation energy and of electrons, migra-tion of molecules and chemical reacmigra-tions P700: reaction

center of photosystem I; P680: reaction center of

photo-system II; LHC: light-harvesting complex; Q: quinones; PC, plastocyanin; Fd: ferredoxin; cyt: cytochromes

2.2 Supply and Demand of CO2

in the Photosynthetic Process

The rate of photosynthetic carbon assimilation is determined by both the supply and demand for CO2 The supply of CO2to the chloroplast is gov-erned by diffusion in the gas and liquid phases and can be limited at several points in the pathway from the air surrounding the leaf to the site of carboxyla-tion inside The demand for CO2is determined by the rate of processing the CO2 in the chloroplast which is governed by the structure and biochemis-try of the chloroplast (Sect 2.1), by environmental factors such as irradiance, and factors that affect plant demand for carbohydrates (Sect 4.2) Limita-tions imposed by either supply or demand can determine the overall rate of carbon assimilation, as explained below

2.2.1 Demand for CO2—the CO2-Response

Curve

The response of photosynthetic rate to CO2 concen-tration is the principal tool to analyze the demand for CO2 and partition the limitations imposed by demand and supply (Warren 2007, Flexas et al 2008) (Fig 6) The graph giving net CO2assimilation (An) as a function of CO2concentration at the site of Rubisco in the chloroplast (Cc) is referred to as the An -Cccurve With rising CO2, there is no net CO2 assim-ilation, until the production of CO2 in respiration (mainly photorespiration, but also some dark respira-tion occurring in the light) is fully compensated by the fixation of CO2in photosynthesis The CO2 con-centration at which this is reached is the CO2 -com-pensation point () In C3 plants this is largely determined by the kinetic properties of Rubisco,

FIGURE Schematic representation of the photosyn-thetic carbon reduction cycle (Calvin cycle) showing major steps: carbon fixation, triose-P production and regeneration of RuBP 1: CO2 combines with its

sub-strate, ribulose-1,5-bisphosphate (RuBP), catalyzed by ribulose bisphosphate carboxylase/oxygenase (Rubisco), producing phosphoglyceric acid (PGA) 2: PGA is reduced to triose-phosphate (triose-P), in a

two-step reaction; the reaction for which ATP is required is the conversion of PGA to 1,3-bisphosphogly-cerate, catalyzed by phosphoglycerate kinase and 4: Part of the triose-P is exported to the cytosol, in exchange for Pi; the remainder is used to regenerate

FIGURE5 Reactions and organelles involved in

photore-spiration In C3plants, at 20% O2, 0.035% CO2, and 208C,

two out of ten RuBP molecules are oxygenated, rather than carboxylated The oxygenation reaction produces phos-phoglycolate (GLL-P), which is dephosphorylated to glyco-late (GLL) Glycoglyco-late is subsequently metabolized in peroxisomes and mitochondria, in which glyoxylate

(GLX) and the amino acids glycine (GLY) and serine (SER) play a role Serine is exported from the mitochondria and converted to hydroxypyruvate (OH-PYR) and then glyce-rate (GLR) in the peroxisomes, after which it returns to the chloroplast (after Ogren 1984) Reprinted with kind per-mission from the Annual Review of Plant Physiology, Vol 35, copyright 1984, by Annual Reviews Inc

with values for  in the range 40—50 mmol (CO2) mol—1 (air) (at 258C and atmospheric pressure)

Two regions of the CO2-response curve above the compensation point can be distinguished At low Cc, that is below values normally found in leaves (approximately 165 mmol mol—1), photosynthesis increases steeply with increasing CO2 concentra-tion This is the region where CO2limits the rate of functioning of Rubisco, whereas RuBP is present in saturating quantities (RuBP-saturated or CO2 -limited region) This part of the An—Ccrelationship is also referred to as the initial slope or the carbox-ylation efficiency At light saturation and with a fully activated enzyme (Sect 3.4.2 for details on ‘‘activation’’), the initial slope governs the carboxy-lation capacity of the leaf which in turn depends on the amount of active Rubisco

In the region at high Cc, the increase in Anwith increasing Cclevels off CO2no longer restricts the carboxylation reaction, but now the rate at which RuBP becomes available limits the activity of Rubisco (RuBP-limited region) This rate, in turn, depends on the activity of the Calvin cycle, which ultimately depends on the rate at which ATP and

NADPH are produced in the light reactions; in this region, photosynthetic rates are limited by the rate of electron transport This may be due to limitation by light or, at light saturation, by a limited capacity of electron transport (Box 2A.1) Even at a high Cc, in the region where the rate of electron transport, J, no longer increases with increasing Cc, the rate of net CO2 assimilation continues to increase slightly, because the oxygenation reaction of Rubisco is increasingly suppressed with increasing CO2 con-centration, in favor of the carboxylation reaction At a normal atmospheric concentration of CO2(Ca) and O2(ca 380 and 210000 mmol mol—1, respectively) and at a temperature of 208C, the ratio between the car-boxylation and oxygenation reaction is about 4:1 How exactly this ratio and various other parameters of the An—Cc curve can be assessed is further explained in Box 2A.1 Typically, plants operate at a Ccwhere CO2and electron transport co-limit the rate of CO2assimilation (i.e., the point where the Rubisco-limited/saturated and the RuBP-limited part of the CO2-response curve intersect) This allows effective utilization of all components of the light and dark reactions

FIGURE6 The relationship between the rate of net CO2

assimilation (An) and the CO2 concentration at the

site of Rubisco in the chloroplasts (Cc) for a C3leaf: the

‘‘demand function’’ The concentration at which An¼0

is the CO2-compensation point () The rate of diffusion

of CO2from the atmosphere to the intercellular spaces

and to Rubisco in the chloroplast is given by the ‘‘supply functions’’ (the red and blue lines) The slopes of these lines are the leaf conductance (gL) and mesophyll

conductance (gm), respectively The intersection of the

‘‘supply functions’’ with the ‘‘demand function’’ is the actual rate of net CO2assimilation at a value of Ciand Cc

that occurs in the leaf intercellular spaces (Ci) and at

the site of Rubisco (Cc) for Cain normal air (indicated by

the vertical line) The difference in Andescribed by the

Box 2A.1

Modeling C3Photosynthesisis

Based on known biochemical characteristics of Rubisco and the requirement of NADPH2 and ATP for CO2assimilation, Farquhar et al (1980) developed a model of photosynthesis in C3plants This model was recently updated, based on the CO2 concentration in the chloroplast (Cc) rather than the intercellular CO2 concentration (Ci) (Sharkey et al 2007) It is widely used in ecophy-siological research and more recently also in glo-bal change modeling The model elegantly demonstrates that basic principles of the biochem-istry of photosynthesis explain physiological properties of photosynthesis of intact leaves

Net CO2assimilation (An) is the result of the rate of carboxylation (Vc) minus photorespiration and other respiratory processes In photorespira-tion, one CO2molecule is produced per two oxy-genation reactions (Vo) (Fig 5) The rate of dark respiration during photosynthesis may differ from dark respiration at night, and is called ‘‘day respiration’’ (Rday):

An¼ Vc 0:5Vo Rday (1)

CO2-limited and O2-limited rates of carboxy-lation and oxygenation are described with stan-dard Michaelis—Menten kinetics When both substrates are present, however, they

competi-tively inhibit each other An effective Michaelis— Menten constant for the carboxylation reaction (Km) that takes into account competitive inhibi-tion by O2is described as

Kmẳ Kc1 ỵ O=KoÞ (2)

where Kcand Koare the Michaelis—Menten con-stants for the carboxylation and oxygenation reaction, respectively, and O is the oxygen concentration

The rate of carboxylation in the CO2-limited part of the CO2-response curve (Fig 1) can then be described as

VcẳVcmax  Cc

Ccỵ Km (3)

where Vcmax is the rate of CO2 assimilation at saturating Cc (note that the subscript ‘‘max’’ refers to the rate at saturating Cc)

The ratio of oxygenation and carboxylation depends on the specificity of Rubisco for CO2 relative to O2(Sc/o) which varies widely among photosynthetic organisms (Von Caemmerer 2000), but much less so among C3higher plants (Galme´s et al 2005) Increasing temperature, however, decreases the specificity, because Ko decreases faster with increasing temperature than Kcdoes (Fig 35)

continued

FIGURE The response of net

photo-synthesis (An) to the CO2concentration

in the chloroplast (Cc) at 258C and light

saturation (solid black line) Calcula-tions were made as explained in the text, with values for Vcmax, Jmax, and

Rday of 90, 117, and mmol m–2 s–1,

respectively The lower part of the An

-Ccrelationship (Ac; red line) is limited

by the carboxylation capacity (Vcmax)

and the upper part (Aj; green line) by

the electron-transport capacity (Jmax;

blue line) The rate of electron transport (J/4; blue line) is also shown

Box 2A.1Continued

The CO2-compensation point in the absence of Rday(*) depends on the specificity factor and the O2concentration (O):

ẳ 0:5 O=Sc=o Sc=Soị (4)

* increases more strongly with rising tem-perature than would be expected from the decrease in Sc/obecause the solubility in water for CO2(Sc) decreases more with increasing tem-perature than does that for O2(So) * shows little variation among C3angiosperms as follows from the similarity of Sc/o * is determined experi-mentally and used to calculate the ratio of carbox-ylation and oxygenation as dependent on CO2:

Vo=Vc¼ 2=Cc (5)

thus avoiding the need for incorporating the spe-cificity factor and solubilities (Equation 4)

In the RuBP-limited part of the CO2-response curve (Fig 1.1), the rate of electron transport (J) is constant Increasing Ccincreases the rate of car-boxylation at the expense of the rate of oxygena-tion There is a minimum requirement of four electrons per carboxylation or oxygenation reac-tion Hence, the minimum electron transport rate (J) required for particular rates of carboxylation and oxygenation is

J ẳ4Vcỵ Voị (6)

At light saturation, J is limited by the capacity of electron transport and is called Jmax

Using Equations (5) and (6), the rate of carbox-ylation can then be expressed as

Vcẳ J=f41 ỵ 2=Ccịg (7)

The CO2-limited and RuBP-saturated rate of photosynthesis (Ac) can then be calculated using Equations (1), (3), and (5) as

Ac¼VcmaxðCc  ị Ccỵ Km

 Rday (8)

The RuBP-limited rate of photosynthesis (Aj) can be calculated using Equations (1), (5), and (7) as

Ajẳ JCc  ị

4Ccỵ 2ị Rday (9)

The minimum of Equations (8) and (9) describes the full CO2-response curve as shown in Fig

In the above equations, gas concentrations are expressed as molar fraction (mol mol—1) If required, partial pressure can be converted to molar fraction by dividing it by total air pressure The CO2conductance for CO2diffusion in the mesophyll (gm) can only be calculated when the concentration in the chloroplast (Cc) is known gmcan then be calculated from Cias

An=gm¼ Ci Cc (10)

Information about gm may not always be available As an approximation, the same model can be used assuming that Cc¼ Ci Parameter values specific for that scenario should then be used (see below)

Parameter values for the above equations are normally given for 258C Values for other tem-peratures can be calculated from their tempera-ture dependencies, as described by the generic equation:

parameter ẳ exp c  DH=R TLị (11)

Where TLis leaf temperature (K), R is the molar gas constant, c is a dimensionless constant, and DH is the activation energy (kJ mol—1) Para-meter values estimated for Nicotiana tabacum(to-bacco) for the CO2 response at 258C, an atmospheric pressure, of 99.1 kPa, and an infinite (Cc¼ Ci) and a finite gm, together with the tem-perature dependencies for the latter scenario (Bernacchi et al 2001, 2002) are

Cc¼Ci

finite gm

(Cc<Ci)

CO2response

parameter at 258C at 258C c DHa

* (mmol mol1)

42.75 37.43 19.02 24.46

Kc(mmol

mol1)

404.9 272.4 38.28 80.99

Ko(mmol

mol1)

278.4 165.8 14.68 23.72

Temperature dependencies of model para-meters describing the rates of metabolic pro-cesses that are leaf specific (Jmax, Vmax, Rday) are calculated in a similar manner, but Equation (11) must then be multiplied by the rates at 258C For constants and activation energies, see Bernacchi et al (2001, 2003)

2.2.2 Supply of CO2—Stomatal and Boundary

Layer Conductances

The supply of CO2by way of its diffusion from the surrounding atmosphere to the intercellular spaces (this CO2concentration is denoted as Ci) and to the site of carboxylation in the chloroplasts (this CO2 concentration is described as Cc) represents a limita-tion for the rate of photosynthesis The magnitude of the limitation can be read from the An—Cccurve as the difference in photosynthetic rate at Caand Ciand Cc, respectively (Fig 6) To analyze diffusion limita-tions it is convenient to use the term resistance, because resistances can be summed to arrive at the total resistance for the pathway When considering fluxes, however, it is more convenient to use con-ductance, which is the reciprocal of resistance, because the flux varies in proportion to the conductance

In a steady state, the rate of net CO2assimilation (An) equals the rate of CO2diffusion into the leaf The rate of CO2diffusion can be described by Ficks first law Hence:

Anẳ gcCa Ccị ẳ Ca CcÞ=rc (1)

where, gcis the leaf conductance for CO2transport; Caand Ccare the mole or volume fractions of CO2in air at the site of carboxylation and in air, respec-tively; rcis the inverse of gc(i.e., the leaf resistance for CO2transport)

The leaf conductance for CO2transport, gc, can be derived from measurements on leaf transpiration, which can also be described by Fick’s first law in a similar way:

E ¼ gwwi waị ẳ wi waịrw (2)

where gw is the leaf conductance for water vapor transport; wiand waare the mole or volume frac-tions of water vapor in air in the intercellular spaces and in air, respectively; rwis the inverse of gw(i.e., the leaf resistance for water vapor transport); and E is the rate of leaf transpiration E can be measured directly The water vapor concentration in the leaf can be calculated from measurements of the leaf’s

temperature, assuming a saturated water vapor pressure inside the leaf Under most conditions this is a valid assumption Therefore, the leaf con-ductance for water vapor transport can be determined

The total leaf resistance for water vapor transfer, rw, is largely composed of two components that are in series: the boundary layer resistance, ra, and the stomatal resistance, rs The boundary layer is the thin layer of air adjacent to the leaf that is modified by the leaf (Fig in Chapter 4A on the plant’s energy balance) Turbulence is greatly reduced there, and transport is largely via diffusion Its limit is commonly defined as the point at which the properties of the air are 99% of the values in ambient air The boundary layer resistance can be estimated by measuring the rate of evaporation from a water-saturated piece of filter paper of exactly the same shape and size as that of the leaf Conditions that affect the boundary layer, such as wind speed, should be identical to those during measurements of the leaf resistance The stomatal resistance for water vapor transfer (rs) can now be calculated since rwand raare known:

rwẳ raỵ rs (3)

The resistance for CO2 transport (rc) across boundary layer and stomata can be calculated from rw, taking into account that the diffusion coef-ficients of the two molecules differ The ratio H2O diffusion/CO2diffusion in air is approximately 1.6, because water is smaller and diffuses more rapidly than CO2 This value pertains only to the movement of CO2inside the leaf air spaces and through the stomata For the boundary layer above the leaf, where both turbulence and diffusion influence flux, the ratio is approximately 1.37

rc¼ ra:1:37ị ỵ rs:1:6ị ẳ 1=gc (4)

Cican now be calculated from Equation (1), after substitution of Cifor Cc If calculated according to this, Ciis the CO2concentration at the point where evaporation occurs inside the leaf (i.e., largely the mesophyll cell walls bordering the substomatal

Box 2A.1Continued

Values of Ccdepend on the balance between supply and demand for CO2 The demand func-tion is described above; the supply funcfunc-tion is described in Sect 2.2.2 Electron-transport rates depend on irradiance (Sect 3.2.1), where the

equation describing net CO2 assimilation as a function of irradiance can be used to calculate J by substituting J and Jmax for An and Amax, respectively A combination of these mathemati-cal equations makes it possible to model C3 photosynthesis over a wide range of environ-mental conditions

cavity), but higher than Cc, the CO2concentration at the point where Rubisco assimilates CO2 (Sect 2.2.3)

In C3plants, Ciis generally maintained at around 250 mmol mol—1, but may increase to higher values at a low irradiance and higher humidity of the air, and decrease to lower values at high irradiance, low water availability, and low air humidity For C4 plants, Ciis around 100 mmol mol—1(Osmond et al 1982)

Under most conditions, the stomatal conduc-tance is considerably less than the boundary layer conductance (ga is up to 10 mol m—2s—1, at wind speeds of up to m s—1; gs has values of up to mol m—2s—1at high stomatal density and widely open stomata), so that stomatal conductance strongly influences CO2diffusion into the leaf For large leaves in still humid air, where the boundary layer is thick, however, the situation is opposite

2.2.3 The Mesophyll Conductance

For the transport of CO2from the substomatal cavity to the chloroplast, a mesophyll conductance (also called internal conductance), gm(or resistance, rm)

should be considered Hence, we can describe the net rate of net CO2assimilation by

An ẳ Ca Ccị=raỵ rsỵ rmị (5)

Until fairly recently, the mesophyll conductance has been assumed to be large and has often been ignored in analyses of gas-exchange measurements However, recent evidence shows that this is not justified (Warren 2007, Flexas et al 2008) In addi-tion, we have come to realize that gmchanges with environmental conditions, and often quite rapidly, compared with changes in stomatal conductance (Flexas et al 2007a, Warren 2007)

Two types of measurements are commonly employed for the estimation of Cc, which is subse-quently used to calculate gm Carbon-isotope fractionation(Box 2A.2) during gas exchange, and simultaneous measurement of chlorophyll fluores-cenceand gas exchange The two methods rely on a number of assumptions that are largely indepen-dent, but they yield similar results (Evans & Loreto 2000) From the estimates made so far, it appears that gmis of similar magnitude as gs; whilst gmis generally somewhat higher, the opposite can also be observed (Galme´s et al 2007) Consequently, Ccis

Box 2A.2

Fractionation of Carbon Isotopes in Plants

CO2in the Earth’s atmosphere is composed of different carbon isotopes The majority is12CO2; approximately 1% of the total amount of CO2in the atmosphere is13CO2; a much smaller fraction is the radioactive species14CO2(which will not be dealt with in the present context) Modern ecophysiological research makes abundant use of the fact that the isotope composition of plant biomass differs from that of the atmosphere Carbon isotopes are a crucial tool in estimating time-integrated measures of photosynthetic per-formance of individual plants or plant commu-nities, information that would be difficult or impossible to obtain from direct physiological measurements It is of special interest that carbon-isotope composition differs among plants that differ in photosynthetic pathway or water-use efficiency How can we account for that?

The molar abundance ratio, R, of the two car-bon isotopes is the ratio between13C and12C The

constants K12 and K13 refer to the rate of pro-cesses and reactions in which12C and13C parti-cipate, respectively The ‘‘isotope effect’’ is described as

Rsource=Rproduct¼ k12=k13 (1)

For plants, the isotope effect is, to a small extent, due to the slower diffusion in air of 13CO

2, when compared with that of the lighter isotope12CO2(1.0044 times slower; during diffu-sion in water, there is little fractionation) (Table 1) The isotope effect is largely due to the biochemical properties of Rubisco, which reacts more readily with12CO

2than it does with13CO2 As a result, Rubisco discriminates against the heavy isotope For Rubisco from Spinacia olera-cea(spinach), the discrimination is 30.3%, whereas smaller values are found for this enzyme from bacteria (Guy et al 1993)

Box 2A.2Continued

TABLE The magnitude of fractionation during CO2uptake

Process or enzyme Fractionation (%)

Diffusion in air 4.4 Diffusion through the boundary

layer

2.9

Dissolution of CO2 1.1

Diffusion of aqueous CO2 0.7

CO2and HCO3in –8.5 at 308C

equilibrium –9.0 at 258C CO2- HCO3catalyzed by

carbonic anhydrase

1.1 at 258C

HCO3- CO2in water,

catalyzed by carbonic anhydrase

10.1 at 258C

PEP carboxylase 2.2 Combined process –5.2 at 308C

–5.7 at 258C

Rubisco 30 at 258C

Source: Henderson et al 1992

On the path from intercellular spaces to Rubisco a number of additional steps take place, where some isotope fractionation can occur Taken together, the isotope effect in C3 plants is approximated by the empirical equation (Farquhar et al 1982):

Ra=Rpẳ 1:0044 ẵCa Ciị=Ca ỵ 1:027 Ci=Ca (2)

where Raand Rpare the molar abundance ratios of the atmospheric CO2and of the C fixed by the plant, respectively; the symbols Caand Ciare the atmospheric and the intercellular partial pres-sure of CO2, respectively The value 1.027 is an empirical value, incorporating the major fractio-nation by Rubisco, as well as accounting for the internal diffusion resistance for CO2(gm)

Since values for Ra/Rp appear rather ‘‘clumsy,’’ data are commonly expressed as frac-tionation values, D (‘‘capital delta’’), defined as (Ra/Rp— 1)  1000, or:

Dẳ ẵ1:0044 Ca 1:0044 Ciỵ 1:027 Ciị=Ca 

ẳ ẵ1:0044 Caỵ 0:0226 Ciị=Ca 

ẳ 4:4 ỵ 22:6 Ci=Caị  103

(3)

The isotope composition is described as 13C (‘‘lower case delta’’):

13C %oị ẳ Rsample=Rstandard 1ị  1000 (4)

Values for D13C and 13C are related as

D¼ source plantị= ỵ plantị (5)

where source 8% if the source is air (air) (to be entered as 0.008 in Equation (5); a pl value of 27%, therefore, converts to a D value of 19.5%) The standard is a cretaceous limestone consisting mostly of the fossil carbonate skele-tons of Belemnitella americana (referred to as PDB-belemnite) By definition, it has a 13C value equal to 0% Plant 13C values are negative, because they are depleted in13C compared with the fossil standard Diffusion and carboxylation discriminate against 13CO

2; -values for C3 plants are approx 27%, showing that Rubisco is the predominant factor accounting for the observed values and that diffusion is less important

For C4plants, the following empirical equa-tion has been derived:

Dẳ 4:4 ỵ ẵ5:7 ỵ 30  1:8ịf  4:4 Ci=Ca (6)

where f refers to the leakage of CO2from the bundle sheath to the mesophyll

Where these equations lead us? Within C3 plants the 13C of whole-plant biomass gives a better indication of Ci over a longer time interval than can readily be obtained from gas-exchange measurements The value of Ciin itself is a reflection of stomatal con-ductance (gs), relative to photosynthetic activ-ity (A) As such, 13C provides information on a plant’s water-use efficiency (WUE) (Sect 5.2) How we arrive there? As can be derived from Equation (3), the extent of the fractionation of carbon isotopes depends on the intercellular partial pressures of CO2, relative to that in the atmosphere If Ciis high, gsis large relative to A, and much of the13CO2 discriminated against by Rubisco diffuses back to the atmosphere; hence the fractiona-tion is large If Ciis low, then relatively more of the accumulated13CO2is fixed by Rubisco, and therefore the fractionation of the overall photosynthesis process is less Comparison of WUE calculated on the basis of 13C is only valid at constant vapor pressure differ-ence (Dw) and is called intrinsic WUE (A/gs)

continued

substantially lower than Ci(the CO2concentration in the intercellular spaces); a difference of about 80 mmol mol—1is common, as compared with Ca—Ci of about 100 mmol mol—1 The mesophyll conductance varies widely among species and correlates with the photosynthetic capacity (Amax) of the leaf (Fig 7) Interestingly, the relationship between mesophyll conductance and photosynthesis is rather similar for scleromorphic and mesophytic leaves, but scler-omorphs tend to have a somewhat larger draw-down of CO2between intercellular space and chloroplast (Ci—Cc) (Warren & Adams 2006)

The mesophyll conductance is a complicated trait, involving diffusion of CO2in the intercellular spaces in the gas phase, dissolving of CO2in the liquid phase, conversion of CO2into HCO3 cata-lyzed by carbonic anhydrase, and diffusion in the

liquid phase and across membranes The resistance in the gas phase is low and is considered as normally not a limiting factor (Bernacchi et al 2002) Diffusion in the liquid phase is much slower (104times less), and the path length is minimized by chloroplast position against the cell wall opposite intercellular spaces (Fig 1) This component likely represents a large fraction of total rm, and carbonic anhydrase is important for minimizing it (Gillon & Yakir 2000) Evidence for an important role for the area of chlor-oplasts bordering intercellular spaces as a determi-nant of gmstems from a positive relationship with this parameter per unit leaf area (Evans & Loreto 2000) Data about a similar parameter, chloroplast area per leaf area, are more widely available and vary by an order of magnitude among species (Table 1) which is likely associated with gm There

Box 2A.2Continued

Under many situations 13C is a good proxy for WUE and it can be used for, e.g., paleoclimatic studies and genetic screening for drought-toler-ant varieties However, under conditions where Dwvaries or gs and gmare not strongly corre-lated, 13C may not be a good predictor of WUE Carbon-isotope fractionation values differ between C3, C4, and CAM species (Sects and 10) In C4plants, little of the13CO2that is discri-minated against by Rubisco diffuses back to the atmosphere This is prevented, first, by the diffu-sion barrier between the vascular bundle sheath and the mesophyll cells Second, the mesophyll cells contain PEP carboxylase, which scavenges most of the CO2 that escapes from the bundle sheath (Table 1) Fractionation during photo-synthesis in C4plants is therefore dominated by fractionation during diffusion (4.4%) There is also little fractionation in CAM plants, where the heavy isotopes discriminated against cannot readily diffuse out of the leaves because the sto-mata are closed for most of the day The actual 13C of CAM plant biomass depends on the frac-tions of the carbon fixed by CAM and C3 photosynthesis

Aquatic plants show relatively little fractiona-tion, due to unstirred layers surrounding the leaf, rather than to a different photosynthetic path-way (Sect 11.6) The unstirred boundary layers cause diffusion to be a major limitation for their photosynthesis, so that fractionation in these

plants tends toward the value found for the dif-fusion process (Fig 1)

FIGURE1 The relationship between the ratio of the

internal and the atmospheric CO2concentration, at a

constant Caof 340 mmol mol–1 Data for both C3and

C4species are presented; the lines are drawn on the

basis of a number of assumptions, relating to the extent of leakage of CO2 from the bundle sheath

is evidence that specific aquaporins facilitate trans-port of CO2 across membranes Their role in the transport of CO2might account for rapid modula-tion of gmin response to environmental factors such as temperature, CO2, and desiccation (Flexas et al 2006a) The mesophyll conductance is proportional to chloroplast surface area within a given functional group The difference in gm between functional groups is associated with mesophyll cell wall

thickness, which varies from 0.1 mm in annuals, 0.2—0.3 mm in deciduous, broad-leaved species, and 0.3—0.5 mm in evergreen, broad-leaved species (Terashima et al 2006)

When stomatal and mesophyll conductance are considered in conjunction with the assimilation of CO2, the ‘‘supply function’’ (Equation 1) tends to inter-sect the ‘‘demand function’’ in the region where carbox-ylation and electron transport are co-limiting (Fig 6)

TABLE The area of the chloroplast in palisade (P) and spongy (S) mesophyll (Areachlor) expressed per unit leaf area (Arealeaf) for species from the mountain range of the East Pamirs, Tadjikistan (3500–4500 m).*

(Areachlor) /Arealeaf

P S PỵS Lowest (PỵS) Highest (PỵS)

Perennial dicotyledonous herbs (54) 12 18 41 Cushion plants (4) 20 11 26 12 40 Dwarf semishrubs (12) 16 21 48

Subshrubs (8) 15 24

Source: Pyankov & Kondratchuk (1995, 1998)

*

The number of investigated species is given in brackets The sum P+S differs from PỵS, because data pertain to both dorsiventral (PỵS) and isopalisade (P) species

FIGURE7 The relationship between the rate of

photo-synthesis (An) and maximum mesophyll conductance

(gm), determined for a wide range of species Values

for scleromorphic leaves are at most 0.21 mol m–2s–1 bar–1(gm) and 22.9 mmol m–2s–1(An), whereas those for

mesomorphic leaves span the entire range shown here The units of conductance as used in this graph differ from those used elsewhere in this text The reason is

that when CO2is dissolving to reach the sites of

carbox-ylation, the amount depends on the partial pressure of CO2and conductance has the units used in this graph

For air space conductance the units could be the same as used elsewhere: mol m–2s–1, if CO

2is given as a mole

fraction (based on data compiled in Flexas et al 2008) Courtesy, J Flexas, Universitat de les Illes Balears, Palma de Mallorca, Balears, Spain

3 Response of Photosynthesis to Light

The level of irradiance is an important ecological factor on which all photo-autotrophic plants depend Only the photosynthetically active part of the spectrum (PAR; 400—700 nm) directly drives photosynthesis Other effects of radiation pertain to the photoperiod, which triggers flowering and other developmental phenomena in many species, the direction of the light, and the spectral quality, characterized by the red/far-red ratio, which is of major importance for many aspects of morphogen-esis These effects are discussed in Chapter on growth and allocation and Chapter on life cycles; effects of infrared radiation are discussed in Chapter 4A on the plant’s energy balance and its significance through temperature effects on photosynthesis in Sect Effects of ultraviolet radiation are treated briefly in Sect 2.2 of Chapter 4B on effects of radia-tion and temperature

Low light intensities pose stresses on plants because irradiance limits photosynthesis and thus net carbon gain and plant growth Responses of the photosynthetic apparatus to shade can be at two levels: either at the structural level, or at the level of the biochemistry in chloroplasts Leaf anatomy, and structure and biochemistry of the photosyn-thetic apparatus are treated in Sect 3.2.2; aspects of morphology at the whole plant level are discussed in Sect 5.1 of Chapter on growth and allocation

High light intensities may also be a stress for plants, causing damage to the photosynthetic appa-ratus, particularly if other factors are not optimal The kind of damage to the photosynthetic apparatus that may occur and the mechanisms of plants to cope with excess irradiance are treated in Sect 3.3

To analyze the response of photosynthesis to irradiance, we distinguish between the dynamic response of photosynthesis to light (or any other environmental factor) and the steady-state response A steady-state response is achieved after exposure of a leaf to constant irradiance for some time until a constant response is reached Dynamic responses are the result of perturbations of steady-state conditions due to sudden changes in light con-ditions resulting in changes in photosynthetic rates Certain genotypes have characteristics that are adaptive in a shady environment (shade-adapted plants) In addition, all plants have the capability to acclimate to a shady environment, to a greater or lesser extent, and form a shade plant phenotype (shade form) The term shade plant may therefore refer to an ‘‘adapted’’ genotype or an ‘‘acclimated’’

phenotype Similarly, the term sun plant normally refers to a plant grown in high-light conditions, but is also used to indicate a shade-avoiding species or ecotype The terms sun leaf and shade leaf are used more consistently; they refer to leaves that have developed at high and low irradiance, respectively

3.1 The Light Climate Under a Leaf Canopy

The average irradiance decreases exponentially through the plant canopy, with the extent of light attenuation depending on both the amount and arrangement of leaves (Monsi & Saeki 1953, 2005):

I ¼ IoekL (6)

where I is the irradiance beneath the canopy; Iois the irradiance at the top of the canopy; k is the extinction coefficient; and L is the leaf area index (total leaf area per unit ground area) The extinction coefficient is low for vertically inclined leaves (for example 0.3—0.5 for grasses), higher for a more horizontal leaf arrangement, and approaching 1.0 for ran-domly distributed, small, perfectly horizontal leaves A clumped leaf arrangement and deviating leaf angles result in intermediary values for k A low extinction coefficient allows more effective light transfer through canopies dominated by these plants Leaves are more vertically inclined in high-light than in cloudy or shaded environments This minimizes the probability of photoinhibition and increases light penetration to lower leaves in high-light environments, thereby maximizing whole-canopy photosynthesis (Terashima & Hikosaka 1995) Values for leaf area index range from less than in sparsely vegetated communities like deserts or tundra to 5—7 for crops to 5—10 for forests (Schulze et al 1994)

The spectral composition of shade light differs from that above a canopy, due to the selective absorption of photosynthetically active radiation by leaves Transmittance of photosynthetically active radiation is typically less than 10%, whereas transmittance of far-red (FR, 730 nm) light is sub-stantial (Fig in Chapter on life cycles) As a result, the ratio of red (R, 660 nm) to far-red (the R/FR ratio) is lower in canopy shade This affects the photoequilibrium of phytochrome, a pigment that allows a plant to perceive shading by other plants (Box 7.2), and requires adjustment of the photosynthetic apparatus

So there are short spells of high irradiance against a background of a low irradiance Such sunflecks are due to the flutter of leaves, movement of branches and the changing angle of the sun Their duration ranges from less than a second to minutes Sunflecks typically have lower irradiance than direct sunlight due to penumbral effects, but large sunflecks (those greater than an angular size of 0.5 degrees) can approach irradiances of direct sunlight (Chazdon & Pearcy 1991)

3.2 Physiological, Biochemical, and Anatomical Differences Between Sun and Shade Leaves

Shade leaves exhibit a number of traits that make them quite distinct from leaves that developed in full daylight We first discuss these traits and then some of the problems that may arise in leaves from exposure to high irradiance In the last section we discuss signals and transduction pathways that allow the formation of sun vs shade leaves

3.2.1 The Light-Response Curve of Sun and Shade Leaves

The steady-state rate of CO2assimilation increases asymptotically with increasing irradiance Below the light-compensation point (An ¼ 0), there is insufficient light to compensate for respiratory car-bon loss due to photorespiration and dark respira-tion (Fig 8) At low light intensities, Anincreases linearly with irradiance, with the light-driven elec-tron transport limiting photosynthesis The initial slope of the light-response curve based on absorbed light (quantum yield) describes the efficiency with which light is converted into fixed carbon (typically about 0.06 moles CO2 fixed per mole of quanta under favorable conditions and a normal atmo-spheric CO2 concentration) When the light-response curve is based on incident light, the leaf’s absorptance also determines the quantum yield; this initial slope is called the apparent quantum yield At high irradiance, photosynthesis becomes light-saturated and is limited by carboxylation rate, which is governed by some combination of CO2 diffusion into the leaf and carboxylation capacity The shape of the light-response curve can be satis-factorily described by a nonrectangular hyperbola (Fig 9):

AnẳfI ỵ Amax

pffI ỵ Amaxị2  fmaxg

2  Rd (7)

where Amaxis the light-saturated rate of gross CO2 assimilation (net rate of CO2 assimilation ỵ dark respiration) at infinitely high irradiance,  is the (apparent) quantum yield (on the basis of either incident or absorbed photons),  is the curvature factor, which can vary between and 1, and Rdis the dark respiration during photosynthesis The Equa-tion can also be used to describe the light depen-dence of electron transport, when A is then replaced by J and Amaxby Jmax(Box 2A.1) This mathematical description is useful because it contains variables with a clear physiological meaning that can be derived from light-response curves and used to model photosynthesis

Sun leaves differ from shade leaves primarily in their higher light-saturated rates of photosynthesis (Amax) (Fig 9) The rate of dark respiration typically covaries with Amax The initial slope of the light-response curves of light-acclimated and shade-accli-mated plants (the quantum yield) is the same, except when shade-adapted plants become inhib-ited or damaged at high irradiance (photoinhibition or photodestruction) which reduces the quantum yield The apparent quantum yield (i.e., based on incident photon irradiance) may also vary with var-iation in absorptance due to differences in chloro-phyll concentration per unit leaf area This is typically not important in the case of acclimation to light (Sect 3.2.3), but cannot be ignored when factors such as nutrient availability and senescence

FIGURE8 Typical response of net photosynthesis to

irra-diance, drawn according to Equation (7) in the text The intercept with the x-axis is the light-compensation point (LCP), the initial slope of the line gives the quantum yield (f) and the intercept with the y-axis is the rate of dark respiration (Rd) The curvature of the line is described by

q At low irradiance, the rate of CO2assimilation is

light-limited; at higher irradiance Anis carboxylation limited

Amaxis the light-saturated rate of CO2assimilation at

ambient Ca

play a role The transition from the light-limited part to the light-saturated plateau is generally abrupt in shade leaves, but more gradual in sun leaves (higher Amaxand lower  in sun leaves) Although shade leaves typically have a low Amax, they have lower light-compensation points and higher rates of

photosynthesis at low light because of their lower respiration rates per unit leaf area (Fig 9)

Just as in acclimation, most plants that have evolved under conditions of high light have higher light-saturated rates of photosynthesis (Amax), higher light-compensation points, and lower rates

FIGURE9 Photosynthesis as a function of irradiance for

different species and growing conditions Light acclima-tion: (A) for Atriplex triangularis (Bj ăorkman 1981) and (B) for a thin algal culture (Coccomyxa sp.) grown at different levels of irradiance 100, 400, or 600 mmol m–2 s–1(B) note the difference in ‘‘curvature’’, for which the

of photosynthesis at low light than shade-adapted plants when grown under the same conditions

3.2.2 Anatomy and Ultrastructure of Sun and Shade Leaves

One mechanism by which sun-grown plants, or sun leaves on a plant, achieve a high Amax(Fig 9) is by producing thicker leaves (Fig 10) which provides space for more chloroplasts per unit leaf area The increased thickness is largely due to the formation of longer palisade cells in the mesophyll and, in spe-cies that have this capacity, the development of multiple palisade layers in sun leaves (Hanson 1917) Plants that naturally occur in high-light envir-onments (e.g., grasses, Eucalyptus and Hakea species) may have palisade parenchyma on both sides of the leaf (Fig 10) Such leaves are naturally positioned (almost) vertically, so that both sides of the leaf receive a high irradiance Anatomy constrains the potential of leaves to acclimate, e.g., the acclimation potential of shade leaves to a high-light environ-ment is limited by the space in mesophyll cells bor-dering intercellular spaces (Oguchi et al 2005) Full acclimation to a new light environment, therefore, typically requires the production of new leaves

The spongy mesophyll in dorsiventral leaves of dicotyledons increases the path length of light in

leaves by reflection at the gas-liquid interfaces of these irregularly oriented cells The relatively large proportion of spongy mesophyll in shade leaves therefore enhances leaf absorptance, due to the greater internal light scattering (Vogelmann et al 1996) When air spaces of shade leaves of Hydrophyl-lum canadense (broad-leaved waterleaf) or Asarum canadense (Canadian wild-ginger) are infiltrated with mineral oil to eliminate this phenomenon, light absorptance at 550 and 750 nm is reduced by 25 and 30%, respectively (Fig 11) In sun leaves, which have relatively less spongy mesophyll, the effect of infiltration with oil is much smaller The optical path length in leaves ranges from 0.9 to 2.7 times that of an equivalent amount of pigment in water, greatly increasing the effectiveness of light absorption in thin leaves of shade plants (Ruăhle & Wild 1979)

Leaves of obligate shade plants, as can for instance be found in the understory of a tropical rainforest, may have specialized anatomical struc-tures that enhance light absorption even further Epidermal and sub-epidermal cells may act as lenses that concentrate light on chloroplasts in a thin layer of mesophyll

There are fewer chloroplasts per unit area in shade leaves as compared with sun leaves due to the reduced thickness of mesophyll The ultrastruc-ture of the chloroplasts of sun and shade leaves shows distinct differences (Fig 12) Shade

FIGURE10 Light-microscopic trans-verse sections of sun and shade leaves of two species: (Top) Arabi-dopsis thaliana (thale cress) and (Bottom) Chenopodium album (pig-weed) Note that the sun leaves of Arabidopsis thaliana have two cell layers for the palisade tissue while those of Chenopodium album have only one layer Shade leaves of both species have only one cell layer Scale bar ¼ 100 mm (courtesy S Yano, National Institute for Basic Biology, Okazaki, Japan)

FIGURE12 Electron micrographs of

chloroplasts in sun (A–C) and shade (D–F) leaves of Schefflera arboricola (dwarf umbrella plant) Chloroplasts found in upper palisade parenchyma tissue (A, D), lower palisade par-enchyma tissue (B, E) and spongy mesophyll tissue (C, F) Note the dif-ference in grana between sun and shade leaves and between the upper and lower layer inside the leaf Scale bar ¼ 0.2 mm (courtesy A.M Syme and C Critchley, Depart-ment of Botany, The University of Queensland, Australia)

FIGURE11 (A) Light absorptance in a shade leaf of Hydro-phyllum canadense (broad-leaved waterleaf) The solid line gives the absorptance of a control leaf The broken line shows a leaf infiltrated with mineral oil, which reduces light scattering The difference between the two

Box 2A.3

Carbon–Fixation and Light–Absorption Profiles Inside Leaves

We are already familiar with differences in bio-chemistry and physiology between sun and shade leaves (Sect 3.2) If we consider the gradient in the level of irradiance inside a leaf, however, then should we not expect similar differences within leaves? Indeed, palisade mesophyll cells at the adaxial (upper) side of the leaf tend to have char-acteristics associated with acclimation to high irradiance: a high Rubisco/chlorophyll and chl a/chl b ratio, high levels of xanthophyll-cycle carotenoids, and less stacking of the thylakoids (Fig 13; Terashima & Hikosaka 1995) On the other hand, the spongy mesophyll cells at the abaxial (lower) side of the leaf have chloroplasts with a lower Rubisco/chlorophyll and chl a/chl bratio, characteristic for acclimation to low irra-diance What are the consequences of such pro-files within the leaf for the exact location of carbon fixation in the leaf?

To address this question we first need to know the light profile within a leaf which can be mea-sured with a fiberoptic microprobe that is moved through the leaf, taking light readings at different wavelengths (Vogelmann 1993) Chlorophyll is not homogeneously distributed in a cell; rather, it is concentrated in the chloroplasts that may have an heterogeneous distribution within and between cells In addition, inside the leaf, absorp-tion varies because of scattering at the air-liquid interfaces that modify pathlength (e.g., between palisade and spongy mesophyll) (Sect 3.2.4) How can we obtain information on light absorption?

After a period of incubation in the dark, chlorophyll fluorescence of a healthy leaf is pro-portional to the light absorbed by that leaf (Box 2A.4) Vogelmann & Evans (2002) illuminated leaves at the adaxial side and at the side of a transversal cut, and measured the distribution of fluorescence over the cut surface using imaging techniques Fluorescence obtained with adaxial light represents light absorption, whereas lighting the cut surface represents chlorophyll concentra-tion In leaves of Spinacia oleracea (spinach), going from the upper leaf surface deeper into the leaf, the chlorophyll concentration increases to 50 mmol m—2over the first 250 mm in the palisade layer, remains similar deeper down in the palisade and spongy mesophyll, but then declines steeply toward the lower surface over the last 100 mm of

the spongy mesophyll layer (Fig 1A) As expected from the absorption characteristics of chlorophyll (Fig 2), green light is less strongly absorbed than blue and red, penetrates deeper into the leaf, and, consequently, shows there a higher absorption (Fig 1A) The data on light absorption and chlor-ophyll concentration allow the calculation of an extinction coefficient, which varies surprisingly little across a leaf Differences in scattering bet-ween the two mesophyll layers are apparently not very important as is also evident from measurements of infiltrated leaves (Fig 11; Vogelmann & Evans 2002)

What are the consequences of the profiles of absorption and chlorophyll concentration for the distribution of photosynthetic activity across a section of a leaf? The profile of photosynthetic capacity (Amax) can be measured following fixa-tion of 14CO2 ensuring light saturation for all chloroplasts (Evans & Vogelmann 2003); alterna-tively, the profile of Rubisco concentration can be used (Nishio et al 1993) Both techniques require making thin sections parallel to the leaf surface Amaxpeaks where chlorophyll reaches its maxi-mum in the palisade mesophyll, and declines to a lower value in the spongy mesophyll (Fig 1A) Amax per chlorophyll decreases similarly from the palisade to the spongy mesophyll We can use the profiles of Amaxand absorbed irradiance to calculate photosynthetic activity (A) in each layer from the light-response curve, using virtually the same equation as introduced in Sect 3.2.1 (the only difference being that Rdayis left out):

A ẳfI ỵ Amax

 ffI ỵ Amaxị2 4fIAmax

0:5

g

2

(1)

where f is the maximum quantum yield, I is the absorbed irradiance and  describes the curva-ture The calculated light-response curves of the adaxial layers are like those of sun leaves, whereas those of the abaxial layers are like the ones of shade leaves Photosynthetic activity peaks close to the adaxial surface in low light, but the maximum shifts to deeper layers at higher irradiances (Fig 1B) Since green light

continued

chloroplasts have a smaller volume of stroma, where the Calvin-cycle enzymes are located, but larger grana, which contain the major part of the chlorophyll Such differences are found both between plants grown under different light condi-tions and between sun and shade leaves on a single plant, as well as when comparing chloroplasts from the upper and lower side of one, relatively thick, leaf of Schefflera arboricola (dwarf umbrella plant) (Fig 12) The adaxial (upper) regions have a chlor-oplast ultrastructure like sun leaves, whereas

shade acclimation is found in the abaxial (lower) regions of the leaf (Box 2A.3)

3.2.3 Biochemical Differences Between Shade and Sun Leaves

Shade leaves minimize light limitation through increases in capacity for light capture and decreased carboxylation capacity and mesophyll conductance, but this does not invariably lead to higher chloro-phyll concentrations per unit leaf area which

Box 2A.3Continued

has a lower absorptance, A in that spectral region is more homogeneously distributed across the leaf profile, whereas blue light causes a sharp

peak closer to the upper surface Calculated pro-files of A show a close match with the experi-mental data of the14C-fixation profile.

FIGURE Profiles of chlorophyll and

light absorption (A), and photosynthesis (B) in a leaf of Spinacia oleracea (spi-nach) The distribution of chlorophyll was derived from measurements of chlorophyll fluorescence, using a light source to illuminate the cut surface of a transversal section of the leaf The absorption of green and blue light was also measured with chlorophyll fluores-cence, but with light striking the upper leaf surface The light-saturated photo-synthetic electron transport rate (Amax)

determines their absorptance (Terashima et al 2001, Warren et al 2007) Some highly shade-adapted species [e.g., Hedera helix (ivy) in the juvenile stage], however, may have substantially higher chlorophyll levels per unit leaf area in shade This might be due to the fact that their leaves not get much thinner in the shade; however, there may also be some photodestruction of chlorophyll in high light in such species In most species, however, higher levels of chlorophyll per unit fresh mass and per chloroplast in shade leaves are compen-sated for by the smaller number of chloroplasts and a lower fresh mass per area This results in a rather constant chlorophyll level per unit area in sun- and shade leaves

The ratio between chlorophyll a and chlorophyll b (chl a/chl b) is lower in shade-acclimated leaves These leaves have relatively more chlorophyll in the light-harvesting complexes, which contain large amounts of chl b (Lichtenthaler & Babani 2004) The decreased chl a/chl b ratio is therefore a reflection of the greater investment in LHCs (Evans 1988) The larger pro-portion of LHC is located in the larger grana of the shade-acclimated chloroplast (Fig 12) Sun leaves also contain more xanthophyll carotenoids, relative to chlorophyll (Box 2A.3; Lichtenthaler 2007)

Sun leaves have larger amounts of Calvin-cycle enzymes per unit leaf area as compared with shade leaves, due to more cell layers, a larger number of chloroplasts per cell, and a larger volume of stroma, where these enzymes are located, per chloroplast, compared with shade leaves Sun leaves also have more stroma-exposed thylakoid membranes, which contain the b6f cytochromes and ATPase (Fig 13) All these components enhance the photosynthetic capacityof sun leaves Since the amount of chloro-phyll per unit area is more or less equal among leaf types, sun leaves also have a higher photosynthetic capacity per unit chlorophyll The biochemical gra-dients for Rubisco/chlorophyll across a leaf are similar to those observed within a canopy, with adaxial (upper) cells having more Rubisco, but less chlorophyll than abaxial (lower) cells (Terashima & Hikosaka 1995)

3.2.4 The Light-Response Curve of Sun and Shade Leaves Revisited

Table summarizes the differences in characteristics between shade-acclimated and sun-acclimated leaves (Walters 2005) The higher Amaxof sun leaves as compared with shade leaves is associated with a greater amount of compounds that determine

photosynthetic capacity which are located in the greater number of chloroplasts per area and in the larger stroma volume and the stroma-exposed thy-lakoids in chloroplasts The increase of Amaxwith increasing amount of these compounds is almost linear (Evans & Seemann 1989) Hence, investment in compounds determining photosynthetic capacity is proportionally translated into photosynthetic rate at high irradiance levels

The higher rate of dark respiration in sun leaves is not only due to a greater demand for respiratory energy for the maintenance of the larger number of leaf cells and chloroplasts, because respiration rates drop rapidly upon shading, whereas Amaxis still high (Pons & Pearcy 1994) Much of the demand for ATP is probably associated with the export of the products of photosynthesis from the leaf and other processes

FIGURE13 Nitrogen partitioning among various

compo-nents in shade- and sun-acclimated leaves Most of the leaf’s N in herbaceous plants is associated with photo-synthesis Some of the fractions labeled Bios (Biosynth-esis) and Rem (Remainder) are indirectly involved in synthesis and maintenance processes associated with the photosynthetic apparatus LH ¼ light harvesting (LHC, PS I, PS II), ETỵCF ẳ electron transport compo-nents and coupling factor (ATPase), CR ¼ enzymes asso-ciated with carbon reduction (Calvin cycle, mainly Rubisco), Bios ¼ biosynthesis (nucleic acids and ribo-somes), Rem ¼ remainder, other proteins and N-con-taining compounds (e.g., mitochondrial enzymes, amino acids, cell wall proteins, alkaloids) (after Evans & See-mann 1989)

associated with a high photosynthetic activity (Sect 4.4 in Chapter 2B on plant respiration)

The preferential absorption of photons in the red and blue regions of the spectrum by a leaf is not a simple function of its irradiance and chlorophyll concentration A relationship with a negative exponent would be expected, as described for monochromatic light and pigments in solution (Lambert-Beer’s law) The situation in a leaf is more complicated, however, because preferential absorption of red light by chlorophyll causes changes in the spectral distribution of light through the leaf Moreover, the path length of light is com-plicated, due to reflection inside the leaf and to changes in the proportions of direct and diffuse light Empirical equations, such as a hyperbole, can be used to describe light absorption by chlor-ophyll For a healthy leaf, the quantum yield based on incident light is directly proportional to the amount of photons absorbed

The cause of the decrease in convexity (Equation 7) of the light-response curve with increasing growth irradiance (Fig 9) is probably partly associated with the level of light-acclimation of the chloroplast in the cross-section of a leaf in relation to the distribution of light within the leaf (Leverenz 1987)

A high Amaxper unit area and per unit chloro-phyll (but not per unit biomass) of sun leaves is beneficial in high-light conditions, because the pre-vailing high irradiance can be efficiently exploited, and photon absorption per unit photosynthetic capacity is not limiting photosynthetic rates Such a high Amax, however, would not be of much use in the shade, because the high irradiance required to utilize the capacity occurs only infrequently, and a high Amaxis associated with high rates of respiration and a large investment of resources On the other hand, a high chlorophyll concentration per unit photosyn-thetic capacity and per unit biomass in thin shade leaves maximizes the capture of limiting photons in TABLE2 Overview of generalized differences in characteristics

between shade- and sun-acclimated leaves

Sun Shade

Structural

Leaf dry mass per area High Low Leaf thickness Thick Thin Palisade parenchyma thickness Thick Thin Spongy parenchyma thickness Similar Similar Stomatal density High Low Chloroplast per area Many Few Thylakoids per stroma volume Low High Thylakoids per granum Few Many Biochemical

Chlorophyll per chloroplast low high Chlorophyll per area similar similar Chlorophyll per dry mass low high Chlorophyll a/b ratio high low Light-harvesting complex per area low high Electron transport components per area high low Coupling factor (ATPase) per area high low Rubisco per area high low Nitrogen per area high low Xanthophylls per area high low Gas exchange

low-light conditions which is advantageous at low irradiance Apparently, there is a ‘‘trade-off’’ between investment of resources in carbon-assimilating capacity and in light harvesting as reflected in the ratio of photosynthetic capacity to chlorophyll concentration This ratio repre-sents light acclimation at the chloroplast level

Although Amaxper unit chlorophyll responds quali-tatively similar to growth irradiance in all plants, there are differences among species (Fig 14; Murchie & Horton 1997) Four functional groups can be discerned:

1 Shade-avoiding species, such as the pioneer tree Betula pendula(European white birch) have a high Amax/chlorophyll ratio This ratio, however, does not change much with growth irradiance Fast-growing herbaceous species from habitats

with a dense canopy and/or a variable light avail-ability have high Amax/chlorophyll ratios, which decrease strongly with decreasing irradiance Plan-tago lanceolata(snake plantain) and Arabidopsis thali-ana(thale cress) (Bailey et al 2001) are examples A plastic response is also found in shade-adapted

plants such as herbaceous understory species [Alocasia macrorrhiza (giant taro)] that depend on gaps for reproduction, and forest trees that toler-ate shade as seedlings The Amax/chlorophyll ratio, however, is much lower over the entire range of irradiance levels

4 A low Amax/chlorophyll ratio that changes little with growth irradiance is found in woody shade-adapted species, such as juvenile Hedera helix (ivy)

3.2.5 The Regulation of Acclimation

As mentioned in previous sections, light acclimation consists of changes in leaf structure and chloroplast

number at the leaf level, and changes in the photo-synthetic apparatus at the chloroplast level Some aspects of leaf anatomy, including morphology of epidermal cells and the number of stomata, are con-trolled by systemic signals originating in mature leaves (Lake et al 2001, Coupe et al 2006) Chloro-plast properties are mostly determined by the local light environmentof the developing leaves (Yano & Terashima 2001)

Studies of regulation at the chloroplast level have yielded significant insights Each of the major compo-nents of the photosynthetic apparatus has part of their subunits encoded in the chloroplast and others in the nucleus Acclimation of chloroplast composition thus likely entails coordinated changes in transcription of both genomes The abundance of mRNAs coding for photosynthetic proteins, however, does not respond clearly during acclimation which suggests that post-transcriptional modifications play an important role (Walters 2005) Several perception mechanisms of the spectral and irradiance component of the light climate have been proposed

Mutants lacking cryptochrome and phytochrome photoreceptors (CRY1, CRY2, PHYA), or having defects in their signaling pathway, show changes in chloroplast composition and disturbance of normal acclimation (Smith et al 1993, Walters et al 1999) Hence, these photoreceptors are either actually involved in perception of the light environment with respect to photosynthetic acclimation, or their action is a prerequisite for normal development of the photo-synthetic apparatus There is also evidence for a role of signals from photosynthesis itself in the regulation of acclimation, either directly or indirectly Several of these have been identified, such as the redox state of components of the photosynthetic membrane or in the stroma, the ATP/ADP ratio, reactive oxygen species,

FIGURE 14 Light-saturated rate of CO2 assimilation

(Amax) per unit chlorophyll in relation to growth

irra-diance for four different species Plantago lanceolata (snake plantain) (Poot et al 1996), Betula pendula (European white birch, Bp) ( ăOquist et al 1982), Alo-casia macrorrhiza (giant taro, Am) (Sims & Pearcy 1989), Hedera helix (ivy, Hh) (T.L Pons, unpublished data)

and the concentration of carbohydrates, including glu-cose and trehalose-6-phosphate (Walters 2005), but a definitive answer about their precise role is still lack-ing Systemic signals play a role in the effect of the light environment of mature leaves on the acclimation of young, growing leaves, irrespective of their own light environment (Yano & Terashima 2001)

3.3 Effects of Excess Irradiance

All photons absorbed by the photosynthetic pig-ments result in excited chlorophyll, but at irradiance levels beyond the linear, light-limited region of the light-response curve of photosynthesis, not all excited chlorophyll can be used in photochemistry (Figs 8, 15) The fraction of excitation energy that cannot be used increases with irradiance and under conditions that restrict the rate of electron transport and Calvin-cycle activity such as low temperature and desiccation This is potentially harmful for plants, because the excess excitation may result in serious damage, if it is not dissipated To avoid damage, plants have mechanisms to safely dispose of this excess excitation energy When these mechanisms are at work, the quantum yield of photosynthesis is temporarily reduced (minutes), a normal phenomenon at high irradiance The excess excitation energy, however, may also cause damage to the photosynthetic membranes if the dissipation mechanisms are inadequate This is called

photoinhibition, which is due to an imbalance between the rate of photodamage to PS II and the rate of repair of damaged PS II Photodamage is initiated by the direct effects of light on the O2 -evol-ving complex and, thus, photodamage to PS II is unavoidable (Nishiyama et al 2006) A reduction in quantum yield that is re-established within min-utes to normal healthy values is referred to as dynamic photoinhibition(Osmond 1994); it is pre-dominantly associated with changes in the xantho-phyll cycle (Sect 3.3.1) More serious damage that takes hours to revert to control conditions leads to chronic photoinhibition; it is mostly related to tem-porarily impaired D1 (Sect 2.1.1; Long et al 1994) Even longer-lasting photoinhibition (days) can be referred to as sustained photoinhibition (Sect 7.2) A technique used for the quantification of photoinhi-bition is the measurement of quantum yield by means of chlorophyll fluorescence (Box 2A.4)

3.3.1 Photoinhibition—Protection by Carotenoids of the Xanthophyll Cycle

Plants that are acclimated to high light dissipate excess energy through reactions mediated by a par-ticular group of carotenoids (Fig 16) This dissipa-tion process is induced by accumuladissipa-tion of protons in the thylakoid lumen which is triggered by excess light Acidification of the lumen induces an enzy-matic conversion of the carotenoid violaxanthin into antheraxanthin and zeaxanthin (Gilmore 1997) The

Box 2A.4

Chlorophyll Fluorescence

When chlorophyllous tissue is irradiated with photosynthetically active radiation (400—700 nm) or wavelengths shorter than 400 nm, it emits radiation of longer wavelengths (approx 680—760 nm) This fluorescence origi-nates mainly from chlorophyll a associated with photosystem II (PS II) The measurement of the kinetics of chlorophyll fluorescence has been developed into a sensitive tool for probing state variables of the photosynthetic apparatus in vivo In an ecophysiological context, this is a useful technique to quantify effects of stress on photosynthetic performance that is also applic-able under field conditions

Photons absorbed by chlorophyll give rise to (1) an excited state of the pigment which is chan-nelled to the reaction center and may give rise to photochemical charge separation The quantum yield of this process is given by fP Alternative routes for the excitation energy are (2) dissipa-tion as heat (fD) and (3) fluorescent emission (fF) These three processes are competitive This leads to the assumption that the sum of the quantum yields of the three processes is unity:

fPỵ fDỵ fFẳ I (1)

Since only the first two processes are subject to regulation, the magnitude of fluorescence depends on the added rates of photochemistry and heat dissipation Measurement of fluores-cence, therefore, provides a tool for quantifica-tion of these processes

Basic Fluorescence Kinetics

When a leaf is subjected to strong white light after incubation in darkness, a characteristic pattern of fluorescence follows, known as the Kautsky curve (Bolha`r-Nordenkampf & ăOgren 1993, Schreiber et al 1995) It rises immediately to a low value (F0), which is maintained only briefly in strong light, but can be monitored for a longer period in weak intermittent light (Fig 1, left) This level of fluorescence (F0) is indicative of open reaction centers due to a fully oxidized state of the primary electron acceptor QA In strong saturating irradiance, fluorescence

continued

FIGURE1 Fluorescence kinetics in

dark-incubated and illuminated leaves in response to a saturating pulse of white light mod ¼ modulated measuring light on; sat ¼ saturating pulse on; act¼acti-nic light on for a prolonged period together with modulated measuring light; -act ¼ actinic light off For expla-nation of fluorescence symbols see text (after Schreiber et al 1995)

Box 2A.4Continued

rises quickly to a maximum value (Fm) (Fig 1, left) which indicates closure of all reaction cen-ters as a result of fully reduced QA When light is maintained, fluorescence decreases gradually (quenching) to a stable value as a result of induc-tion of photosynthetic electron transport and dis-sipation processes

After a period of illumination at a sub-saturating irradiance, fluorescence stabilizes at a value F, somewhat above F0 (Fig 1, right) When a saturating pulse is given under these conditions, fluorescence does not rise to Fm, but to a lower value called Fm

0

Although reaction centers are closed at saturating light, dissipation processes compete now with fluorescence which causes the quenching of Fmto Fm

0

Since all reaction centers are closed during the saturating pulse, the photochemical quantum yield (fP) is practically zero and, therefore, the quantum yields of dissipation at saturating light (fDm) and fluorescence at saturating light (fFm) are unity:

fDmỵ fFmẳ (2)

It is further assumed that there is no change in the relative quantum yields of dissipation and fluorescence during the saturating pulse:

fDm fFm¼

fD

fF (3)

Photochemical quantum yield (fP) is also referred to as fII because it originates mainly from PSII It can now be expressed in fluores-cence parameters only, on the basis of Equations (1), (2), and (3)

fII¼fFm fF fFm

(4)

The fluorescence parameters F0and Fmcan be measured with time-resolving equipment, where the sample is irradiated in darkness with l5680 nm and fluorescence is detected as emitted radiation at l4680 nm White light sources, however, typically also have radiation in the wavelength region of chlorophyll fluores-cence For measurements in any light condition, systems have been developed that use a weak modulated light source in conjunction with a

detector that monitors only the fluorescence emitted at the frequency and phase of the source A strong white light source for generating satur-ating pulses ð45; 000 mol m2s1Þ and an acti-nic light source typically complete such systems The modulated measuring light is sufficiently weak for measurement of F0 This is the method used in the example given in Fig The constancy of the measuring light means that any change in fluorescence signal is proportional to fF This means that the maximum quantum yield (fIIm) as measured in dark-incubated leaves is

fmẳ Fm F0ị=Fmẳ Fv=Fm (5)

where Fvis the variable fluorescence, the differ-ence between maximal and minimal fluores-cence In illuminated samples the expression becomes

fẳ Fm0 Fị=Fm0ẳ DF=Fm0 (6)

where DF is the increase in fluorescence due to a saturating pulse superimposed on the actinic irradiance DF=F0

m has values equal to or lower than Fv/Fm; the difference increases with increasing irradiance

The partitioning of fluorescence quenching due to photochemical (qP) and nonphotochem-ical (qN) processes can be determined These are defined as

qP ẳ Fm0 Fị=Fm0 F00ị ẳ DF=Fv0 (7)

qN ẳ1  Fm0 F00ị=Fm F0ị ẳ  Fv0=Fv(8)

F0may be quenched in light, and is then called F0

0

(Fig right) The measurement of this para-meter may be complicated, particularly under field conditions We can also use another term for nonphotochemical quenching (NPQ) which does not require the determination of F00:

NPQ ¼ ðFm Fm0Þ=Fm0 (9)

The theoretical derivation of the fluorescence parameters as based on the assumptions described above is supported by substantial empirical evidence The biophysical background of the processes, however, is not always fully understood

Box 2A.4Continued

Relationships with Photosynthetic Performance

Maximum quantum yield after dark incuba-tion (Fv/Fm) is typically very stable at values around 0.8 in healthy leaves Fv/Fm correlates well with the quantum yield of photosynthesis measured as O2 production or CO2 uptake at low irradiance (Fig 2) In particular, the reduc-tion of the quantum yield by photoinhibireduc-tion can be evaluated with this fluorescence para-meter A decrease in Fv/Fm can be due to a decrease in Fm and/or an increase in F0 A fast- and a slow-relaxing component can be distinguished The fast component is alleviated within a few hours of low light or darkness

and is therefore only evident during daytime; it is supposed to be involved in protection of PS II against over-excitation The slow-relaxing component remains several days and is con-sidered as an indication of (longer-lasting) damage to PS II Such damage can be the result of sudden exposure of shade leaves to full sun light, or a combination of high irradi-ance and extreme (high or low) temperature The way plants cope with this combination of stress factors determines their performance in particular habitats where such conditions occur

Quantum yield in light (DF/Fm

0

) can be used to derive the rate of electron transport (JF)

JF¼ I DF=Fm0 abs 0:5 (10)

where I is the irradiance and abs is the photon absorption by the leaf and 0.5 refers to the equal partitioning of photons between the two photosystems (Genty et al 1989) For comparison of JF with photosynthetic gas-exchange rates, the rate of the carboxylation (Vc) and oxygenation (Vo) reaction of Rubisco must be known In C4plants and in C3plants at low O2and/or high CO2, Vo is low and can be ignored Hence, the rate of electron trans-port can also be derived from the rate of O2 production or CO2uptake (Jc) For a compar-ison of JFwith Jcin normal air in C3plants, Vo must be estimated from the intercellular par-tial pressure of CO2 (Box 2A.1) Photosyn-thetic ratesgenerally show good correlations with JF(Fig 3) JFmay be somewhat higher than Jc(Fig 3) This can be ascribed to electron flow associated with nonassimilatory processes, or with assimilatory processes that not result in CO2absorption, such as nitrate reduction Alter-natively, the chloroplast population monitored by fluorescence is not representative for the func-tioning of all chloroplasts across the whole leaf depth The good correlation of gas exchange and fluorescence data in many cases indicates that JF is representative for the whole-leaf photosyn-thetic rate, at least in a relative sense Hence, JF is also referred to as the relative rate of electron transport

continued

FIGURE2 The relationship between quantum yield,

as determined from the rate of O2evolution at

dif-ferent levels of low irradiance, and the maximum quantum yield of PS II determined with chlorophyll fluorescence (Fv/Fm, fIIm) Measurements were

made on Glycine max (soybean) grown at high (open symbols) and low (filled symbols) N supply and exposed to high light for different periods prior to measurement (after Kao & Forseth 1992)

dissipation process also requires a special photosys-tem II subunit S(PsbS) (Li et al 2002) Mutants of Arabidopsis thaliana(thale cress) that are unable to convert violaxanthin to zeaxanthin in excessive light exhibit greatly reduced nonphotochemical quench-ing, and are more sensitive to photoinhibition than wild-type plants (Niyogi et al 1998) Similarly, PsbS-deficient mutants have a reduced fitness at intermittent, moderate levels of excess light (Kuălheim et al 2002)

Zeaxanthin triggers a kind of ‘‘lightning rod’’ mechanism It is involved in the induction of conformational changes in the light-harvesting antennae of PS II which facilitates the dissipation of excess excitations (Fig 17; Pascal et al 2005) This energy dissipation can be measured by chlor-ophyll fluorescence (Box 2A.4) and is termed

high-energy-dependent or pH-dependent fluores-cence quenching In the absence of a properly functioning xanthophyll cycle, excess energy could, among others, be passed on to O2 This leads to photooxidative damage when scaven-ging mechanisms cannot deal with the resulting reactive oxygen species (ROS) For example, her-bicides that inhibit the synthesis of carotenoids cause the production of vast amounts of ROS that cause chlorophyll to bleach and thus kill the plant (Wakabayashi & B ăoger 2002) In the absence of any inhibitors, ROS inhibit the repair of PS II, in particular the synthesis of the D1 proteinof PS II, by their effect on mRNA transla-tion It is a normal phenomenon when plants are exposed to full sunlight even in the absence of other stress factors (Nishiyama et al 2006)

Box 2A.4Continued

FIGURE Relationship of chlorophyll fluorescence

parameters and rates of CO2assimilation in the C3

plant Flaveria pringlei A ¼ rate of CO2assimilation;

fII ¼quantum yield of PS II in light (F/Fm0); JF¼

electron-transport rate calculated from fIIand

irra-diance; Jc¼electron-transport rate calculated from

However, when shade plants are exposed to full sunlight, or when other stresses combine with high irradiance (e.g., desiccation, high or low tem-perature) then more excessive damage can occur, involving destruction of membranes and oxida-tion of chlorophyll (bleaching), causing a longer-lasting reduction in photosynthesis

In sun-exposed sites, diurnal changes in irradi-ance are closely tracked by the level of antherax-anthin and zeaxanthin In shade conditions, sunflecks lead to the rapid appearance of antherax-anthin and zeaxantherax-anthin and reappearance of violax-anthin between subsequent sunflecks This regulation mechanism ensures that no competing dissipation of energy occurs when light is limiting for photosynthesis, whereas damage is prevented when light is absorbed in excess Typically, sun-grown plants not only contain a larger fraction of the carotenoids as zeaxanthin in high light, but their total pool of carotenoids is larger also (Fig 18; Adams et al 1999) The pool of reduced ascorbate, which plays a role in the xanthophyll cycle (Fig 17), is also several-fold greater in plants acclimated to high light (Logan et al 1996)

3.3.2 Chloroplast Movement in Response to Changes in Irradiance

The leaf’s absorptance is affected by the concentra-tion of chlorophyll in the leaf and the path length of light in the leaf, as well as by the location of the chloroplasts Light-induced movements of chloro-plasts are affected only by wavelengths below 500 nm High intensities in this wavelength region cause the chloroplasts to line up along the vertical walls, parallel to the light direction, rather than along the lower cell walls, perpendicular to the direction of the radiation, as in control leaves Chlor-oplasts are anchored with actin networks and their final positioning relies on connections to actin (Stai-ger et al 1997) Chloroplast movements are pro-nounced in aquatic plants, such as Vallisneria gigantea(giant vallis) and shade-tolerant understory species, such as Oxalis oregana (redwood sorrel) where they may decrease the leaf’s absorptance by as much as 20%, thereby increasing both transmit-tance and reflectransmit-tance Other species [e.g., the shade-avoiding Helianthus annuus (sunflower)] show no blue light-induced chloroplast movement or change

FIGURE16 Scheme of the xanthophyll cycle and its

reg-ulation by excess or limiting light Upon exposure to excess light, a rapid stepwise removal (de-epoxidation) of two oxygen functions (the epoxy groups) in violax-anthin takes place; the pH optimum of this reaction, which is catalyzed a epoxidase, is acidic This de-epoxidation results in a lengthening of the conjugated

system of double bonds from in violaxanthin 10 and 11 in antheraxanthin and zeaxanthin, respectively The de-epoxidation step occurs in minutes Under low-light conditions, the opposite process, epoxidation, takes place It may take minutes, hours, or days, depending on environmental conditions (Demmig-Adams & Adams 1996, 2006)

in absorptance Chloroplast movements in shade plants exposed to high light avoid photoinhibition (Brugnoli & Bj ăorkman 1992)

3.4 Responses to Variable Irradiance

So far we have discussed mostly steady-state responses to light, meaning that a particular envir-onmental condition is maintained until a constant response is achieved Conditions in the real world,

however, are typically not constant, irradiance being the most rapidly varying environmental factor Since photosynthesis primarily depends on ance, the dynamic response to variation in irradi-ance deserves particular attention

The irradiance level above a leaf canopy changes with time of day and with cloud cover, often by more than an order of magnitude within seconds In a leaf canopy, irradiance, particularly direct radiation, changes even more In a forest, direct sunlight may penetrate through holes in

FIGURE17 Top: Depiction of the conditions where (A)

all or (B) only part of the sunlight absorbed by chloro-phyll within a leaf is used for photosynthesis Safe dis-sipation of excess energy requires the presence of zeaxanthin as well as a low pH in the photosynthetic membranes The same energized form of chlorophyll is used either for photosynthesis or loses its energy as heat Bottom: Depiction of the regulation of the biochemistry of the xanthophyll cycle as well as the induction of xanthophyll-cycle-dependent energy dissipation by pH De-epoxidation to antheraxanthin (A) and

the overlying leaf canopy, casting sunflecks on the forest floor These move with wind action and position of the sun, thus exposing both leaves in the canopy and shade plants in the understory to short periods of bright light Sun-flecks typically account for 40—60% of total irra-diance in understory canopies of dense tropical and temperate forests and are quite variable in duration and intensity (Chazdon & Pearcy 1991)

3.4.1 Photosynthetic Induction

When a leaf that has been in darkness or low light for hours is transferred to a saturating level of

irradiance, the photosynthetic rate increases only gradually over a period of up to one hour to a new steady-state rate (Fig 19), with stomatal conduc-tance increasing more or less in parallel We cannot conclude, however, that limitation of photosynth-esis during induction is invariably due to stomatal opening (Allen & Pearcy 2000) If stomatal conduc-tance limited photosynthesis, the intercellular CO2 concentration (Ci) should drop immediately upon transfer to high irradiance, but, in fact, there is a more gradual decline over the first minutes, and then a slow increase until full induction (Fig 19) Stomatal patchiness might play a role (Sect 5.1), but there are also additional limitations at the chloro-plast level The demand for CO2 increases faster than the supply in the first minutes as evident from the initial decline in Ci Its subsequent rise indicates that the supply increases faster than the demand, as shown in Fig 20, where Anis plotted as a function of Ci during photosynthetic induction During the first one or two minutes there is a fast increase in demand for CO2(fast induction compo-nent) which is due to fast light induction of some Calvin-cycle enzymes and build-up of metabolite pools (Sassenrath-Cole et al 1994) The slower phase of increase in demand until approximately 10 minutes is dominated by the light-activation of Rubisco After that, Ciincreases and An increases along the An—Cicurve, indicating that a decrease in stomatal limitation dominates further rise in photo-synthetic rate (Fig 20)

Loss of photosynthetic induction occurs in low light, but at a lower rate than induction in high light, particularly in forest understory species Hence, in a sequence of sunflecks, photosynthetic induction increases from one sunfleck to the next, until a high induction state is reached, when sunflecks can be used efficiently (Fig 19)

3.4.2 Light Activation of Rubisco

Rubisco, as well as other enzymes of the Calvin cycle, are activated by light, before they have a high catalytic activity (Fig 21; Portis 2003) The increase in Rubisco activity, due to its activation by light, closely matches the increased photosynthetic rate at a high irradiance apart from possible stoma-tal limitations Two mechanisms are involved in the activation of Rubisco Firstly, CO2binds covalently to a lysine residue at the enzyme’s active site (car-bamylation), followed by binding of Mg2+ and RuBP In this activated state, Rubisco is able to cat-alyze the reaction with CO2or O2 Rubisco is deac-tivatedwhen (1) RuBP binds to a decarbamylated

FIGURE18 Differences in zeaxanthin (Z), violaxanthin

(V) and antheraxanthin (A) contents of leaves upon acclimation to the light level [Vinca minor (periwinkle)], season [Pseudotsuga menziesii (Douglas fir)] and N sup-ply [Spinacia oleracea (spinach)] The total areas reflect the concentration of the three carotenoids relative to that of chlorophyll (after Demmig-Adams & Adams 1996)

Rubisco, (2) 2-carboxy-D-arabinitol 1-phosphate (CA1P), an analogue of the extremely short-lived intermediate of the RuBP carboxylation reaction,

binds to the carbamylated Rubisco, and (3) a product produced by the catalytic ‘‘misfire’’ of Rubisco (‘‘mis-protonation’’), xylulose-1,5-bisphosphate (Salvucci & Crafts-Brandner 2004a), binds to a carbamylated Rubisco Secondly, Rubisco activase plays a role in catalyzing the dissociation of inhibitors from the active site of Rubisco; its activity increases with increasing rate of electron transport (Fig 21) The activity of Rubisco activase is regulated by ADP/ ATP and redox changes mediated by thioredoxin in some species

Light activation of Rubisco, a natural process that occurs at the beginning of the light period in all plants, is an important aspect of the regula-tion (fine-tuning) of photosynthesis In the absence of such light activation, the three phases of the Calvin cycle (carboxylation, reduction, and regeneration of RuBP) may compete for sub-strates, leading to oscillation of the rate of CO2 fixation upon the beginning of the light period It may also protect active sites of Rubisco during inactivity in darkness (Portis 2003), but the reg-ulation mechanism occurs at the expense of low rates of CO2 assimilation during periods of low induction

FIGURE20 Photosynthetic response of Alocasia

macro-rrhiza (giant taro) to intercellular CO2 concentration

(Ci) during the induction phase after a transition from

an irradiance level of 10 to 500 mmol m–2 s–1 (light saturation) The solid line represents the An–Ci

relation-ship of a fully induced leaf calculated as Rubisco-limited rates Numbers indicate minutes after transition (after Kirschbaum & Pearcy 1988)

FIGURE19 Photosynthetic induction in Toona australis,

which is an understory species from the tropical rain-forest in Australia (Left panels) Time course of the rate of CO2assimilation (An) (top), stomatal conductance

(gs) (middle), and the intercellular CO2 concentration

(Ci) (bottom) of plants that were first exposed to a low

3.4.3 Post-illumination CO2Assimilation

and Sunfleck-Utilization Efficiency

The rate of O2 evolution, the product of the first step of electron transport, stops immediately after a sunfleck, whereas CO2assimilation continues for a brief period thereafter This is called post-illu-mination CO2fixation(Fig 22) CO2assimilation in the Calvin cycle requires both NADPH and ATP, which are generated during the light reactions Particularly in short sunflecks, this post-illumina-tion CO2 fixation is important relative to photo-synthesis during the sunfleck, thus increasing total CO2 assimilation due to the sunfleck above what would be expected from steady-state measure-ments (Fig 23)

CO2assimilation due to a sunfleck also depends on induction state Leaves become increasingly induced with longer sunflecks of up to a few

minutes (Fig 25) At low induction states, sun-fleck-utilization efficiency decreases below what would be expected from steady-state rates (Fig 23) Forest understory plants tend to utilize sun-flecks more efficiently than plants from short vege-tation, particularly flecks of a few seconds to a few minutes Accumulation of larger Calvin-cycle meta-bolite pools and longer maintenance of photosyn-thetic induction are possible reasons Efficient utilization of sunflecks is crucial for understory plants, since most radiation comes in the form of relatively long-lasting sunflecks, and half the plant’s assimilation may depend on these short periods of high irradiance

3.4.4 Metabolite Pools in Sun and Shade Leaves

As explained in Sect 2.1.3, the photophosphoryla-tion of ADP depends on the proton gradient across

FIGURE 22 CO2 uptake and

O2 release in response to a

‘‘sunfleck’’ Arrows indicate the beginning and end of the ‘‘sunfleck’’ (Pearcy 1990) With kind permission from the Annual Review of Plant Physiology Plant Molecular Biology, Vol 41, copyright 1990, by Annual Reviews Inc

FIGURE21 (A) Light activation of Rubisco and two other Calvin cycle enzymes, Ribulose-5-phosphate kinase and fructose-bisphosphatase (Salvucci 1989) Copyright Physiologia Plantarum (B) Time course of Rubisco

activation level during sequential light open symbols) and dark (filled symbols) periods (after Portis et al 1986) Copyright American Society of Plant Biologists

the thylakoid membrane This gradient is still pre-sent immediately following a sunfleck, and ATP can therefore still be generated for a brief period The formation of NADPH, however, directly depends on the flux of electrons from water, via the photo-systems and the photosynthetic electron-transport chain, and therefore comes to an immediate halt after the sunfleck Moreover, the concentration of NADPH in the cell is too low to sustain Calvin-cycle activity Storage of the reducing equivalents takes place in triose-phosphates (Table 3), which are intermediates of the Calvin cycle

To allow the storage of reducing power in inter-mediates of the Calvin cycle, the phosphorylating step leading to the substrate for the reduction reac-tion must proceed This can be realized by regulat-ing the activity of two enzymes of the Calvin cycle which both utilize ATP: phosphoglycerate kinase and ribulose-phosphate kinase (Fig 4) When com-peting for ATP in vitro, the second kinase tends to dominate, leaving little ATP for phosphoglycerate kinase If this were to happen in vivo as well, no storage of reducing equivalents in triose-phos-phate would be possible, and CO2 assimilation would not continue beyond the sunfleck The con-centration of triose-phosphate at the end of a

FIGURE23 Efficiency of ‘‘sunfleck’’ utilization as

depen-dent on duration of the ‘‘sunfleck’’ and induction state in two species Alocasia macrorrhiza (giant taro, an understory species) measured at high (closed symbols) and low induction state (open symbols) Induction state of Glycine max (soybean, a sun species) is approximately 50% of maximum Efficiencies are calculated as total CO2 assimilation due to the sunfleck relative to that

calculated from the steady-state rates at the high irradi-ance (sunfleck) and the low (background) irradiirradi-ance (Pearcy 1988, Pons & Pearcy 1992)

TABLE3 The potential contribution of triose-phosphates and ribulose-1,5-bispho-sphate to the post-illumination CO2assimilation of Alocasia macrorrhiza (giant taro) and Phaseolus vulgaris (common bean), grown either in full sun or in the shade.*

Alocasia macrorrhiza Phaseolus vulgaris

Shade Sun Shade Sun

RuBP (mmol m–2)

2.0 14.5 2.9 5.3

Triose phosphates (mmol m–2)

16.3 18.0 19.8 10.5

Total potential CO2fixation

(mmol m–2)

12 25 15 12

Potential efficiency (%)

190 204 154 120

Triose-P/RuBP 4.9 0.7 4.1 1.2

Post-illumination ATP required (mmol g–1Chl)

13 22 63 29

Source: Sharkey et al (1986a)

*The values for the intermediates give the difference in their pool size at the end of the

lightfleck and later The total potential CO2assimilation is RuP2ỵ3/5 triose-P pool

sunfleck is relatively greater in shade leaves than in sun leaves, whereas the opposite is found for ribulose-1,5-bisphosphate (Table 3) This indicates that the activity of the steps in the Calvin cycle leading to ribulose-phosphate is suppressed Thus competition for ATP between the kinase is pre-vented, and the reducing power from NADPH can be transferred to 1,3-bisphosphoglycerate, leading to the formation of triose-phosphate Sto-rage of reducing power occurs in species that are adapted to shade, e.g., Alocasia macrorrhiza (giant taro), as well as in leaves acclimated to shade, e.g., shade leaves of common bean (Phaseolus vulgaris)

3.4.5 Net Effect of Sunflecks on Carbon Gain and Growth

Although most understory plants can maintain a positive carbon balance with diffuse light in the absence of sunflecks, daily carbon assimilation and growth rate in moist forests correlates closely with irradiance received in sunflecks (Fig 24) Moreover, sunflecks account for an increasing proportion of total carbon gain (9—46%) as their size and frequency increase In dry forests, where understory plants experience both light and water limitation, sun-flecks may reduce daily carbon gain on cloud-free days (Allen & Pearcy 2000) Thus, the net impact of

sunflecks on carbon gain depends on both cumula-tive irradiance and other potentially limiting factors

4 Partitioning of the Products of Photosynthesis and Regulation by ‘‘Feedback’’

4.1 Partitioning Within the Cell

Most of the products of photosynthesis are exported out of the chloroplast to the cytosol as triose-phos-phate in exchange for Pi Triose-phosphate is the substrate for the synthesis of sucrose in the cytosol (Fig 25) and for the formation of cellular compo-nents in the source leaf Sucrose is largely exported to other parts (sinks) of the plant, via the phloem

Partitioning of the products of the Calvin cycle within the cell is controlled by the concentration of Piin the cytosol If this concentration is high, rapid exchange for triose-phosphate allows export of most of the products of the Calvin cycle If the concentra-tion of Pidrops, the exchange rate will decline, and the concentration of triose-phosphate in the chloro-plast increases Inside the chlorochloro-plasts, the triose-phosphates are used for the synthesis of starch, releasing Piwithin the chloroplast So, the partition-ing of the products of photosynthesis between export to the cytosol and storage compounds in the chloroplasts is largely determined by the availabil-ity of Piin the cytosol This regulation can be demon-strated by experiments using leaf discs in which the concentration of cytosolic Pi is manipulated (Table 4)

In intact plants the rate of photosynthesis may also be reduced when the plant’s demand for carbo-hydrate (reduced sink strength) is decreased, for example by the removal of part of the fruits or ‘‘girdling’’ of the petiole (Table 4) [Girdling involves damaging the phloem tissue of the stem, either by a temperature treatment or mechanically, leaving the xylem intact.] Restricting the export of assimilates by reduced sink capacity or more directly by block-ing the phloem sequesters the cytosolic Piin phos-phorylated sugars, leading to feedback inhibition of photosynthesis When the level of Piin the cytosol is increased, by floating the leaf discs on a phos-phate buffer, the rate of photosynthesis may also drop [e.g., in Cucumis sativus (cucumber) Table 4], but there is no accumulation of starch This is likely due to the very rapid export of triose-phosphate from the chloroplasts, in exchange for Pi, depleting the Calvin cycle of intermediates

FIGURE24 (A) Total carbon gain of Adenocaulon bicolor

as a function of daily photon flux contributed by sun-flecks in the understorey of a temperate redwood forest (B) Relative growth rate of Euphorbia forbesii (filled circles) and Claoxylon sandwicense (open circles) as a function of average duration of potential sunflecks (esti-mated from hemispherical photographs) in the under-storey of a tropical forest (after Chazdon & Pearcy 1991)

4.2 Short-Term Regulation

of Photosynthetic Rate by Feedback

Under conditions of ‘‘feedback inhibition’’ (Sect 4.1), phosphorylated intermediates of the pathway lead-ing to sucrose accumulate, inexorably decreaslead-ing the cytosolic Piconcentration In the absence of suffi-cient Piin the chloroplast, the formation of ATP is reduced and the activity of the Calvin cycle declines That is, less intermediates are available and less

RuBP is regenerated, so that the carboxylating activ-ity of Rubisco and hence the rate of photosynthesis drops

How important is feedback inhibition in plants whose sink has not been manipulated? To answer this question, we can determine the O2sensitivityof photosynthesis Normally, the rate of net CO2 assimilation increases when the O2concentration is lowered from a normal 21% to or 2%, due to the suppression of the oxygenation reaction When the

FIGURE25 The formation of

triose-phosphate in the Calvin cycle Triose-P is exported to the cytosol, in exchange for inorganic phos-phate (Pi), or used as a substrate

activity of Rubisco is restricted by the regeneration of RuBP, lowering the O2concentration enhances the net rate of CO2assimilation to a lesser extent Feed-back inhibition is found at a high irradiance and also at a low temperature, which restricts phloem load-ing Under these conditions the capacity to assim-ilate CO2exceeds the capacity to export and further metabolize the products of photosynthesis Conse-quently, phosphorylated intermediates of the path-way from triose-phosphate to sucrose accumulate which sequesters phosphate As a result, Pistarts to limit photosynthesis, and the rate of photosynthesis declines as soon as the capacity to channel triose-phosphate to starch is saturated Figure 26, showing the response of the net rate of CO2assimilation to N2 ỵ CO2 at four levels of irradiance and a leaf temperature of 158C, illustrates this point

The assessment of feedback inhibition of photo-synthesis using the O2sensitivity of this process is complicated by the fact that the relative activities of the carboxylating and oxygenating reactions of Rubisco also depend on temperature (Sect 7.1) To resolve this problem, a mathematical model of photosynthesis has been used (Box 2A.1) This model incorporates biochemical information on the photosynthetic reactions and simulates the effect of

TABLE4 Rates of CO2assimilation (mmol m–2s–1) and the accumulation of14C in soluble sugars (‘‘ethanol-soluble’’) and starch (‘‘HClO4-soluble’’) (14C as % of total14C recovered) in leaf discs of Gossypium hirsutum (cotton) and Cucumis sativus (cucumber) floating on a Tris-maleate buffer, a phosphate buffer, or a mannose solution.*

Control Girdled

CO2 Ethanol- HClO4- CO2 Ethanol- HClO4

Fixation Soluble Soluble Fixation Soluble Soluble

Cotton

Tris-maleate 18 83 17 12 76 24

Phosphate 18 87 13 10 83 17

Mannose 12 54 46 10 76 24

Cucumber

Tris-maleate 13 76 24 40 60

Phosphate 82 18 76 24

Mannose 55 45 40 60

Source: Plaut et al (1987)

*

Leaves were taken from control plants (‘‘control’’) or from plants whose petioles had been treated in such a way as to restrict phloem transport (‘‘girdled’’) The concentration of cytosolic Pican be decreased by incubating leaf discs in a

solution containing mannose Mannose is readily taken up and enzymatically converted into mannose phosphate, thus sequestering some of the Pioriginally present in the cytosol Under these conditions starch accumulates in the chloroplasts

At extremely low cytosolic Piconcentrations, the rate of photosynthesis is also reduced When leaf discs are taken from

plants with reduced sink capacity, the addition of mannose has very little effect, because the cytosolic Piconcentration is

already low before mannose addition

FIGURE26 The response of the CO2assimilation rate to a

change in O2concentration at four levels of irradiance

The broken lines give the steady-state rate of CO2

assim-ilation The gas phase changes from air to N2ỵCO2at

the time indicated by the arrows The CO2

concentra-tion in the atmosphere surrounding the leaf is main-tained at 550 mmol mol–1and the leaf temperature at 15oC At a relatively low irradiance (340 mmol m–2s–1) the rate of CO2assimilation is rapidly enhanced when

the O2concentration is decreased, whereas at high

irra-diance (880 mmol m–2 s–1), CO

2 assimilation first

decreases and is only marginally enhanced after several minutes, indicative of feedback inhibition (after Sharkey et al 1986b) Copyright American Society of Plant Biologists

FIGURE27 The effect of temperature on the net rate of

CO2assimilation at 18% (v/v; filled symbols) and 3%

(v/v; open symbols) O2(top), and the O2sensitivity of

lowering the O2concentration at a range of tempera-tures Comparison of the observed effect of the decrease in O2concentration (Fig 27, lower middle and right panels) with the experimental observa-tions (Fig 27, lower left panel), allows conclusions on the extent of feedback inhibition in plants under normal conditions The lower right panels show distinct feedback inhibition for Solanum lycopersicum (tomato) and Populus fremontii (Fremont cotton-wood) at low temperatures, and less feedback inhi-bition for Phaseolus vulgaris (common bean), Capsicum annuum (pepper), Scrophularia desertorum (figwort), and Cardaria draba (hoary cress)

Comparison of the modeled results in the lower left panel with the experimental results in the other lower panels in Fig 27 shows that photo-synthesis of plants growing under natural condi-tions can be restricted by feedback, especially at relatively low temperatures Feedback inhibition is predominantly associated with species accumulat-ing starch in their chloroplasts, rather than sucrose and hexoses in the cytosol and vacuoles Since genetically transformed plants of the same species, lacking the ability to store starch, behave like the starch-accumulating wild type, the reason for this difference remains obscure (Goldschmidt & Huber 1992) Perhaps it reflects the mode of phloem load-ing (i.e., either symplastic or apoplastic; Chapter 2C on long-distance transport)

4.3 Sugar-Induced Repression of Genes Encoding Calvin-Cycle Enzymes

The feedback mechanism outlined in Sect 4.2 oper-ates in the short term, adjusting the activity of the existing photosynthetic apparatus to the capacity of export and sink activity, but mechanisms at the level of gene transcription play a more important role in the long term They modify photosynthetic capacity and can override regulation by light, tissue type, and developmental stage (Smeekens & Rook 1998) Leaves of Triticum aestivum (wheat) fed with 1% glucose have a lower photosynthetic capacity as well as lower levels of mRNA coding for several Calvin-cycle enzymes, including the small subunit of Rubisco (Jones et al 1996) Regulation of photo-synthetic gene expression by carbohydrates plays an important role in the control of the activity of the ‘‘source’’ (leaves) by the demand in the ‘‘sink’’ (e.g., fruits) (Paul & Foyer 2001) Sensing of carbohydrate levels is mediated by a specific hexokinase, which is an enzyme that phosphorylates hexose while hydro-lyzing ATP (Smeekens 2000) This regulation at the level of gene transcription plays a role in the

acclimation of the photosynthetic apparatus to ele-vated concentrations of atmospheric CO2(Sect 12), and, more generally in adjusting photosynthetic capacity to environmental and developmental needs

4.4 Ecological Impacts Mediated by Source-Sink Interactions

Many ecological processes affect photosynthesis through their impact on plant demand for carbohy-drate (Sect 4.2) In general, processes that increase carbohydrate demand increase the rate of photo-synthesis, whereas factors that reduce demand reduce photosynthesis

Although defoliation generally reduces carbon assimilation by the defoliated plant by reducing the biomass of photosynthetic tissue, it may cause a compensatory increase in photosynthetic rate of remaining leaves through several mechanisms The increased sink demand for carbohydrate generally leads to an increase in Amaxin the remaining leaves Defoliation also reduces environmental constraints on photosynthesis by increasing light penetration through the canopy, and by increasing the biomass of roots available to support each remaining leaf The resulting increases in light and water availabil-ity may enhance photosynthesis under shaded and dry conditions, respectively

Growth at elevated atmospheric [CO2] may lead to a down-regulation of photosynthesis, involving sensing of the leaves’ carbohydrate status (Sect 12.1), and other ecological factors discussed here probably act on photosynthesis in a similar manner Box 2A.5 provides a brief overview of gas-exchange equipment, especially portable equip-ment that can be used in the field for ecological surveys

5 Responses to Availability of Water

The inevitable loss of water, when the stomata are open to allow photosynthesis, may lead to a decrease in leaf relative water content (RWC), if the water supply from roots does not match the loss from leaves The decline in RWC may directly or indirectly affect photosynthesis In this section we describe effects of the water supply on photosynth-esis, and discuss genetic adaptation and phenotypic acclimation to water shortage

Box 2A.5

The Measurement of Gas Exchange

The uptake and release of CO2in photosynthesis and respiration and the release of H2O during transpiration of plants or leaves is measured using gas-exchange systems Several types exist (e.g., Field et al 1989, Long & Haăllgren 1993) Here we briefly address the operation of so-called open systems and potential complications with their use, with particular attention to the now commonly used portable systems that are commercially available

The essence of a gas-exchange system is a transparent chamber that encloses the photo-synthetically active tissue Air enters the cham-ber at a specified flow rate (fm) measured and controlled by a flow-controller The leaf changes the concentrations of CO2 and H2O inside the chamber The magnitude of the difference in CO2 and H2O concentration between the air entering the chamber (Ceand We) and at the out-let (Co and Wo) depends on its gas-exchange activity The net photosynthetic rate (An) is then calculated following Von Caemmerer & Farqu-har (1981)

Anẳ fm=LafCe Co1  Weị=1  WoÞg (1)

Anis expressed per unit leaf area (La), but another basis, e.g., dry mass can also be used The last part of the equation refers to the correction for the volume increase and, conse-quently, concentration decrease caused by the simultaneously occurring transpiration (E) E can be calculated similarly by substituting We and Wo for Ceand Co When leaf temperature is also measured, stomatal conductance (gs) can be calculated using E and assuming a saturated vapor pressure in the intercellular spaces of the leaf From gs and An, intercellu-lar CO2 concentration (Ci) can be calculated The principle is explained in Sect 2.2.2, but corrections are necessary (Von Caemmerer & Farquhar 1981) The calculations assume homogeneity of parameter values across the measured area A powerful analysis of photo-synthetic performance can be made when these four gas-exchange parameters are avail-able, as explained in the text A further devel-opment is the combination of gas-exchange

with the measurement of chlorophyll

fluorescence (Box 2A.4) that gives a measure of electron-transport rate allowing estimates of the CO2 concentration in the chloroplast (Cc), conductance for CO2 transport in the mesophyll (gm), photorespiration, and engage-ment of alternative electron sinks (Long & Bernacchi 2003)

A typical leaf chamber contains a fan that homogenizes the air which makes Co and Wo representative of the air around the leaf (Caand Wa, respectively) The fan further increases the boundary layer conductance (Chapter 4A), which allows a better control of leaf temperature and reduces possible errors associated with the estimation of gs It further contains a sensor for leaf and/or air temperature and a light sensor is attached Most of the recent models of portable systems are also equipped with temperature con-trol Concentrations of CO2and H2O at the inlet and outlet of the leaf chamber are measured with an infra-red gas analyzer (IRGA) The concentra-tion of CO2and H2O can be manipulated in the more advanced models that are also equipped with a light source The systems are completed with computerized control, data-acquisition and data-processing This versatile equipment can be used to measure photosynthetic performance in ambient conditions and for measuring the response of gas-exchange activity to environ-mental factors such as humidity, CO2, tempera-ture and light

The ease of gas-exchange measurement brings the danger of less critical use Some sources of error and guidelines for their avoid-ance are given by Long & Bernacchi (2003); here, the most important problems are addressed Modern systems have small chambers that clamp with gaskets on a leaf, thus limiting the measurement to a part of the leaf This has the advantage that also small leaves can easily be measured and that the condition of homogeneity mentioned above is more easily met However, the use of a small area has its draw-backs In these chambers, a significant part of the leaf is covered by the gasket The leaf area under the gasket continues to respire, and part of the CO2 produced diffuses to the leaf chamber where it

5.1 Regulation of Stomatal Opening

Stomatal opening tends to be regulated such that photosynthesis is approximately co-limited by CO2 diffusion through stomata and light-driven electron transport This is seen in Fig as the inter-section between the line describing the leaf’s con-ductance for CO2transport (supply function) and the A-Cccurve (demand function) A higher con-ductance and higher Cc would only marginally increase CO2assimilation, but would significantly increase transpiration, since transpiration increases linearly with gs, as a result of the constant difference in water vapor concentration between the leaf and the air (wi-wa) (Sect 2.2.2, Fig 28; Sect 5.4.3 of Chapter on plant water relations) At lower con-ductance, water loss declines again linearly with gs; however, Ccalso declines, because the demand for CO2remains the same, and the difference with Ca increases This increased CO2concentration gradi-ent across the stomata counteracts the decrease in gs Hence, photosynthesis declines less than does tran-spiration with decreasing Ccand Ci The result is an increasing water-use efficiency (WUE) (carbon gain per water lost) with decreasing gs Less of the total photosynthetic capacity is used at a low Ccand Ci, however, leading to a reduced photosynthetic N-use efficiency(PNUE) (carbon gain per unit leaf N; Sect 6.1)

Plants tend to reduce stomatal opening under water stress so that WUE is maximized at the expense of PNUE Under limited availability of N, stomata may open further, increasing PNUE at the

Box 2A.5Continued

results in overestimation of respiration rates (Pons & Welschen 2002) In homobaric leaves, air can escape through the intercellular spaces depending on the overpressure in the chamber which complicates matters further (Jahnke & Pieruschka 2006) CO2and H2O can also diffuse through the gasket, and more likely along the interface between gasket and leaf This is parti-cularly important when concentrations inside and outside the chamber are different, such as when measuring a CO2 response and at high humidity in a dry environment (Flexas et al 2007b, Rodeghiero et al 2007) Large errors can be caused by these imperfections, particularly when using small chambers at low gas-exchange rates Suggestions are given in the above-men-tioned publications for minimizing and

correcting these errors, but that is not always straightforward and sometimes not possible

When measuring gas-exchange rates under ambient conditions in the field, glasshouse, or growth chamber, ambient light is attenuated, particularly around the edges Ambient air is often used for such measurements The uptake of CO2results in a decreased CO2concentration in the chamber, causing a further underestima-tion of Ancompared with in situ rates The read-ing for E deviates also from in situ rates due to a chamber climate in terms of humidity, tempera-ture, and turbulence that differs from outside When using short periods of enclosure, gs is probably not affected by the chamber climate Corrections of An and E can be made from assumed or separately measured short-term humidity, temperature, light, and CO2 effects, using measurements of environmental para-meters in undisturbed conditions

FIGURE28 The effect of stomatal conductance (gs) on

the transpiration rate (E, mmol m–2s–1), rate of CO2

assimilation (A, mmol m–2s–1), intercellular CO2

con-centration (Ci, mmol mol–1) and photosynthetic

water-use efficiency (WUE, mmol CO2(mol H2O)–1s–1) as a

function of stomatal conductance Calculations were made assuming a constant leaf temperature of 258C and a negligible boundary layer resistance The arrow indicates gsat the co-limitation point of carboxylation

and electron transport For the calculations, Equations as described in Box 2A.1 and Sect 2.2.2 have been used

expense of WUE (Table 5) This trade-off between efficient use of water or N explains why perennial species from lower-rainfall sites in eastern Australia have higher leaf N concentration, lower light-satu-rated photosynthetic rates at a given leaf N concen-tration, and lower stomatal conductance at a given rate of photosynthesis (implying lower Ci) when compared with similar species from higher-rainfall sites By investing heavily in photosynthetic enzymes, a larger draw-down of Ci is achieved, and a given photosynthetic rate is possible at a lower stomatal conductance The benefit of the strat-egy is that dry-site species reduce water loss at a given rate of photosynthesis, down to levels similar to wet-site species, despite occurring in lower-humidity environments The cost of high leaf N is higher costs incurred by N acquisition and possibly increased herbivory risk (Wright et al 2001)

When a plant is subjected to water stress, stomata tend to close This response is regulated initially by abscisic acid(ABA), a phytohormone that is pro-duced by roots in contact with dry soil and is trans-ported to the leaves (Sect 5.4.1 of Chapter on plant water relations; Box 7.2) There are also effects that are not triggered by ABA arriving from the roots, mediated via ABA produced in the leaf (Holbrook et al 2002, Christmann et al 2005) In addition, both electrical and hydraulic signals control stomatal conductance in response to soil moisture availability (Grams et al 2007) Stomatal conductance may also decline in response to increasing vapor pressure deficit (VPD) of the air (Sect 5.4.3 of Chapter on plant water relations) The result of these regulatory mechanisms is that, in many cases, transpiration is fairly constant over a range of VPDs, and leaf water potential is constant over a range of soil water potentials Water loss is therefore restricted when dry air likely imposes water stress (a feedforward response) or when the plant experiences incipient water stress (a feedback response) In dry

environments these two regulatory mechanisms often cause midday stomatal closure and therefore a decline in photosynthesis (Fig 34 in Chapter on plant water relations)

It was long assumed that stomata respond homo-geneously over the entire leaf; however, leaves of water-stressed plants exposed to14CO2show often a heterogeneous distribution of fixed14C This shows that some stomata close completely (there is no radioactivity close to these stomata), whereas others hardly change their aperture (label is located near these stomata) (Downton et al 1988, Terashima et al 1988) This patchy stomatal closure can also be visualized dynamically and nondestructively with thermal and chlorophyll fluorescence imaging tech-niques (Mott & Buckley 2000); patches with closed stomata are identified by their high temperature and low quantum yield Patchiness of stomatal opening complicates the calculation of Ci (Sect 2.2.2), because the calculation assumes a homogeneous distribution of gas exchange parameters across the leaf lamina

Leaves of plants that reduce stomatal conduc-tance during the middle of the day may only close some of their stomata, while others remain open This nonuniform reaction of stomata may occur only when plants are rapidly exposed to water stress, whereas stomata may respond in a more uni-form manner when the stress is imposed more slowly (Gunasekera & Berkowitz 1992) Stomatal patchiness can also occur in dark-adjusted leaves upon exposure to bright light (Eckstein et al 1996, Mott & Buckley 2000)

5.2 The A–CcCurve as Affected

by Water Stress

Water stress alters both the supply and the demand functions of photosynthesis (Flexas & Medrano TABLE5 Intrinsic water-use efficiency (WUE, A/gs) and nitrogen-use efficiency of photosynthesis (PNUE, A/NLA) of leaves of Helianthus annuus (sunflower), growing in a field in the middle of a hot, dry summer day in California.*

NLA A gs Ci WUE PNUE

Mmol m–2 mmol m–2s–1 mol m–2s–1 mmol mol–1 mmol mol1 mmol mol1s1

High N ỵ W 190 37 1.2 240 31 195

Low W 180 25 0.4 200 63 139

Low N 130 27 1.0 260 27 208

Source: Fredeen et al (1991)

*Plants were irrigated and fertilized (high N ỵ W), only irrigated but not fertilized (low N), or only fertilized but not irrigated

2002, Grassi & Magnani 2005), but the main effect is on stomatal and mesophyll conductance, unless the stress is very severe (Fig 29; Flexas et al 2004) When only the conductance declines with plant desiccation, the slope of the An—Cccurve is unaf-fected (Fig 29B) Because high irradiance and high temperature often coincide with drought, however, photoinhibition may be involved which reduces the demand function Similarly, if growth is inhibited more strongly than photosynthesis by water stress, feedback inhibition may play an additional role The net effect of the down-regulation of photosynthetic capacity under severe water stress is that Cc is higher than would be expected if a decrease in con-ductance were the only factor causing a reduction in assimilation in water-stressed plants The reduction in photosynthetic capacity allows photosynthesis to continue operating near the break-point between the RuBP-limited and the CO2-limited regions of the A—Cc curve Thus drought-acclimated plants

maximize the effectiveness of both light and dark reactions of photosynthesis under dry conditions at the cost of reduced photosynthetic capacity under favorable conditions The decline in photosynthetic capacity in water-stressed plants is associated with declines in all biochemical components of the photo-synthetic process

The changes in stomatal regulation of gas exchange in species and cultivars that are geneti-cally adapted to drought are similar to those described above for drought acclimation Drought-adapted wheat (Triticum aestivum) culti-vars have a lower stomatal conductance and oper-ate at a lower Cithan less adapted cultivars In addition, stomatal conductance and photosynth-esis in desert shrubs are lower than in less drought-adapted plants and they decline less in response to water stress, largely due to osmotic adjustment (Sect 4.1 of Chapter on plant water relations)

FIGURE29 The response of net

photosynthesis to (A) intercel-lular CO2 concentration (Ci),

and (B) CO2 concentration in

the chloroplasts (Cc), for well

watered (blue symbols) and severely water-stressed plants (purple symbols) (after Flexas et al 2006b)

5.3 Carbon-Isotope Fractionation in Relation to Water-Use Efficiency

Carbon-isotope composition of plant tissues pro-vides an integrated measure of the photosynthetic water-use efficiency (WUE ¼ A/E) or, more pre-cisely, the intrinsic WUE (A/gs) during the time when the carbon in these tissues was assimilated (Fig 30) As explained in Box 2A.2, air has a 13C of approximately —8%, and the major steps in C3 photo-synthesis that fractionate are diffusion (4.4%) and carboxylation (30%, including dissolution of CO2) The isotopic composition of a leaf will approach that of the process that most strongly limits photosynth-esis If stomata were almost closed and diffusion were the rate-limiting step, 13C of leaves would be about 12.4% (i.e., ỵ 4.4); if carboxylation were the only limiting factor, we would expect a 13C of —38% (i.e., —8 + —30) A typical range of 13C in C

3 plants is —25 to —29%, indicating co-limitation by diffusion and carboxylation (O’Leary 1993); how-ever, 13C values vary among plant species and environment depending on the rate of CO2 assimila-tion and stomatal and mesophyll conductance The fractionation, D13C, is defined as (Box 2A.2):

D13C ¼ ẵ4:4 ỵ 22:6Ci=Caị  103;or :

13Cair 13Cleafẳ 4:4 þ 22:6ðCi=CaÞ; (8)

which indicates that a high Ci/Ca(due to high sto-matal conductance or low rate of CO2assimilation) results in a large fractionation (strongly negative 13C) We can now use this information to estimate an integrated WUE for the plant, but we must be aware of one significant problem: Equation (8) uses

Ci, and does not take mesophyll conductance into account; it uses Ci, and assumes that the mesophyll conductance scales with stomatal conductance Therefore, some of the fractionation data have to be interpreted with great care, because they may reflect differences in mesophyll conductance, rather than (only) stomatal conductance (Grassi & Mag-nani 2005, Warren & Adams 2006)

The water-use efficiency (WUE ¼ An/E) is given by

WUE ẳ An=E ẳ gcCa Ciị=gwwi waị

ẳ Ca1  Ci=Caị=1:6wi waị

(9)

given that gw/gcẳ 1.6 (the molar ratio of diffusion of water vapor and CO2in air) Equation (9) tells us that the WUE is high, if the conductance is low in comparison with the capacity to assimilate CO2in the mesophyll Under these circumstances Ci(and Ci/Ca) will be small The right-hand part of Equa-tion (9) then approximates [Ca/(1.6(wi—wa)] and dif-fusion is the predominant component determining fractionation of carbon isotopes and approaches a value of 4.4% On the other hand, if the stomatal conductance is large, WUE is small, Ciapproximates Ca and the right-hand part of Equation (8) approaches 27% Fractionation of the carbon iso-topes is now largely due to the biochemical fractio-nation by Rubisco Values for WUE thus obtained can only be compared at the same vapor pressure difference (wi—wa), e.g., within one experiment or a site at the same atmospheric conditions Therefore, the WUE derived from 13C of plant carbon is mostly referred to as intrinsic WUE (An/gs), which is equivalent to a value normalized at a constant VPD of mol mol—1.

FIGURE 30 The relationship between carbon-isotope

composition (13C) and (A) average intercellular CO

concentration, and (B) daily photosynthetic water-use efficiency, assimilation/transpiration (A/E) The data

As expected from the theoretical analysis above, there is a good correlation between WUE and the carbon-isotope fractionation (Fig 30) Triticum aesti-vum (wheat) grown under dry conditions has a higher WUE and a lower carbon-isotope fractiona-tion than plants well supplied with water (Farquhar & Richards 1984) Moreover, those genotypes that perform best under drought (greatest WUE) have the lowest carbon-isotope fractionation, so that iso-topic composition can be used to select for

geno-types with improved performance under

conditions where water is limiting (Fig 31) A similar correlation between WUE and 13C has been found for cultivars of other species [e.g., Hor-deum vulgare(barley) (Hubick & Farquhar 1989) and Arachis hypogaea(peanut) (Wright et al 1988, Hubick 1990)]

5.4 Other Sources of Variation in Carbon-Isotope Ratios in C3Plants

Given the close relationship between WUE and 13C, carbon-isotopic composition can be used to infer average WUE during growth (Fig 30; Sect

of Chapter on plant water relations) For example, 13C is higher (less negative) in desert plants than in mesic plants, and it is higher in tissue produced during dry seasons (Smedley et al 1991) or in dry years This indicates that plants growing in dry con-ditions have a lower Cithan those in moist condi-tions Other factors can alter isotopic composition without altering WUE For example, 13C of plant tissue is higher at the bottom than at the top of the canopy This is to a limited extent due to the con-tribution of13C-depleted CO2from soil respiration, but mostly to the lower Ciof sunlit top leaves com-pared with the shaded understory leaves (Buch-mann et al 1997) A complicating factor with the derivation of WUE from 13C is that isotope fractio-nation is operating at the level of Rubisco in the chloroplast, whereas the theoretical model is based on Ci Possible variation in the draw-down of CO2 from the intercellular spaces to the chloroplast (Sect 2.2.3), due to the mesophyll resistance, is not taken into account, and may cause variation in 13C that is not associated with WUE

Annuals fractionate more strongly against 13C than perennials; additionally, herbs fractionate more than grasses, and root parasites [e.g.,

FIGURE31 Association between dry mass and

carbon-isotope fractionation in the F2

genera-tion of Solanum lycopersicum x Solanum pen-nellii (tomato) grown in a wet and a dry environment in 1995 (A) and terminated early and late in 1996 (B) The regression is across the two environments in 1995, whereas in 1996 the regressions are for the early and the late environments separately (Martin et al 1999) Copyright Crop Science Society of America

Comandra umbellata (pale bastard toadflax)] more than any of the surrounding species (Smedley et al 1991) These patterns suggest a high stomatal con-ductance and low WUE in annuals, herbs, and hemi-parasites The low WUE of hemiparasitic plants is important in nutrient acquisition (Sect in Chapter 9D on parasitic associations)

6 Effects of Soil Nutrient Supply on Photosynthesis

6.1 The Photosynthesis–Nitrogen Relationship

Since the photosynthetic machinery accounts for more than half of the N in a leaf (Fig 13) and much of the remainder is indirectly associated with its photosynthetic function, photosynthesis is strongly affected by N availability Amax increases linearly with leaf N per unit area (Fig 32), regardless of whether the variation in leaf N is caused by differences in soil N availability, growth irradiance, or leaf age, and holds also when similar species are compared (Fig 32) The slope of this relationship is much steeper for C4plants than for C3plants (Sect 9.5), and differs also among C3 plants (Sect 4.2.1 of Chapter on mineral nutrition; Evans 1989) When leaves with different N concen-tration are compared of plants grown at different N availability, the photosynthetic rate per unit N (photosynthetic N-use efficiency; PNUE) at the growth irradiance is highest in leaves with low N concentrations This is due to the higher degree of utilization of the photosynthetic apparatus (Fig 33); hence, a higher efficiency at the expense of photosynthetic rate

The strong Amax vs N relationship cannot be due to any simple direct N limitation of photo-synthesis, because both carbon-isotope studies and A—Cccurves generally show that photosynth-esis is co-limited by CO2diffusion and photosyn-thetic capacity Rather, the entire photosynphotosyn-thetic process is down-regulated under conditions of N limitation, with declines in Rubisco, chlorophyll, and stomatal conductance (Sect 5.1, Table 5) The net effect of this coordinated response of all photo-synthetic components is that Ci/Caand 13C show no consistent relationship with leaf N (Rundel & Sharifi 1993)

In some field studies, especially in conifers, which often grow on low-P soils, photosynthesis may show little correlation with tissue N, but a strong correlation with tissue [P] (Reich & Schoettle

1988) The low photosynthetic rate of plants grown at low P supply may reflect feedback inhibition due to slow growth and low concentrations of Piin the cytosol (Sect 4.1) or low concentrations of Rubisco and other photosynthetic enzymes

FIGURE32 The light-saturated rate of photosynthesis

(Amax) of four grasses grown at high (filled symbols)

and low (open symbols) N supply (A) and their photo-synthetic N-use efficiency (PNUE) determined at growth irradiance (B) plotted against leaf N per unit area Note the higher PNUE for plants grown at a low N supply (C) The proportional utilization of the total photosynthetic capacity at growth irradiance, calcu-lated as the ratio of the rate at growth irradiance and Amaxin relation to Amax(Pons et al 1994) Copyright

6.2 Interactions of Nitrogen, Light, and Water

Because of the coordinated responses of all photo-synthetic processes, any environmental stress that reduces photosynthesis will reduce both the diffu-sional and the biochemical components (Table 5) Therefore, N concentration per unit leaf area is typi-cally highest in sun leaves, and declines toward the bottom of a canopy In canopies of Nicotiana tabacum (tobacco), this partially reflects higher rates of CO2 assimilation of young, high-N leaves in high-light environments (Boonman et al 2007) In multi-spe-cies canopies, however, the low leaf [N] per area in understory species clearly reflects the adjustment of photosynthetic capacity to the reduced light avail-ability (Table 5; Niinemets 2007)

6.3 Photosynthesis, Nitrogen, and Leaf Life Span

As discussed in Chapter on mineral nutrition and Chapter on growth and allocation,

plants acclimate and adapt to low soil N and low soil moisture by producing long-lived leaves that are thicker and have a high leaf mass density, a low specific leaf area (SLA; i.e., leaf area per unit leaf mass) and a low leaf N concentration Both broad-leaved and conifer species show a single strong negative correla-tion between leaf life-span and either leaf N concentration or mass-based photosynthetic rate (Fig 33; Reich et al 1997) The low SLA in long-lived leaves relates to structural properties required to withstand unfavorable environmen-tal conditions (Chapter on growth and alloca-tion) There is a strong positive correlation between SLA and leaf N concentration for dif-ferent data sets (Fig 33) Together, the greater leaf thickness and low N concentrations per unit leaf mass result in low rates of photo-synthesis on a leaf-mass basis in long-lived leaves (Fig 33) Maximum stomatal conductance correlates strongly with leaf N, because gsscales with Amax (Wright et al 2004)

FIGURE33 Relations of (A) mass-based maximum rate of

CO2assimilation, (B) leaf N concentration, and (C)

spe-cific leaf area of young mature leaves as a function of

their expected leaf life-span The symbols refer to a data set for 111 species from six biomes (after Reich et al 1997)

7 Photosynthesis and Leaf Temperature: Effects

and Adaptations

Temperature has a major effect on enzymatically catalyzed reactions and membrane processes, and therefore affects photosynthesis Because the activa-tion energyof different reactions often differs among plants acclimated or adapted to different tempera-ture regimes, photosynthesis may be affected accord-ingly (for a discussion of the concepts of acclimation and adaptation, see Fig and Sect of Chapter on assumptions and approaches) In this section tem-perature effects on photosynthesis will be explained in terms of underlying biochemical, biophysical, and molecular processes

Differences among plants in their capacity to per-form at extreme temperatures often correlate with the plant’s capacity to photosynthesize at these tem-peratures This may reflect both the adjustment of photosynthesis to the demand of the sinks (Sect 4) and changes in the photosynthetic machinery dur-ing acclimation and adaptation

7.1 Effects of High Temperatures on Photosynthesis

Many plants exhibit an optimum temperature for photosynthesis close to their normal growth tem-perature, showing acclimation (Fig 34; Berry &

Bjăorkman 1980, Yamori et al 2005) Below this opti-mum, enzymatic reaction rates, primarily associated with the ‘‘dark reactions’’, are temperature limited At high temperatures the oxygenating reaction of Rubisco increases more than the carboxylating one so that photorespiration becomes proportionally more important This is partly because the solubility of CO2 declines with increasing temperature more strongly than does that of O2 Part of the effect of temperature on photosynthesis of C3plants is due to the effects of temperature on kinetic properties of Rubisco Vmaxincreases with increasing temperature, but the Km-values increase also, and more steeply for CO2than for O2(Fig 35) This means that the affinity for CO2decreases more strongly than that for O2 Additionally, electron transport (Cen & Sage 2005) and gm (Yamori et al 2006a, Warren 2007) may decline at elevated temperatures The combined tem-perature effects on solubility, affinity, and mesophyll conductance cause a proportional increase in photo-respiration, resulting in a decline in net photosynth-esis at high temperature when electron-transport rates cannot keep up with the increased inefficiency Adaptationto high temperature typically causes a shift of the temperature optimum for net photo-synthesis to higher temperatures (Fig 36; Berry & Bj ăorkman 1980) Similarly, the temperature opti-mum for photosynthesis shifts to higher tempera-tures when coastal and desert populations of Atriplex lentiformis acclimateto high temperatures (Pearcy 1977)

Apart from the increase in photorespiration dis-cussed above, there are several other factors impor-tant for determining acclimation and adaptation of photosynthesis to temperature In leaves of Spinacia oleracea (spinach) the Rubisco activation state decreases with increasing temperatures above the optimum temperatures for photosynthesis, irrespec-tive of growth temperature, while the activation state remains high at lower temperatures Rubisco ther-mal stabilities of spinach leaves grown at low tem-perature are lower than those of leaves grown at high temperature Photosynthetic performance in spinach is largely determined by the Rubisco kinetics at low temperature and by Rubisco kinetics and Rubisco activation state at high temperature (Yamori et al 2006b) Furthermore, Rubisco can become inacti-vated at moderately high temperatures Species adapted to hot environments often show tempera-ture optima for photosynthesis that are quite close to the temperature at which enzymes are inactivated The lability of Rubisco activase plays a major role in the decline of photosynthesis at high temperatures (Salvucci & Crafts-Brandner 2004b, Hikosaka et al 2006) Thermal acclimation of Acer rubrum (red

FIGURE34 Temperature dependence of light-saturated

maple) from Florida in comparison with genotypes from Minnesota, US, is associated with maintenance of a high ratio of Rubisco activase to Rubisco (Weston et al 2007) In Gossypium hirsutum (cotton) expression of the gene encoding Rubisco activase is influenced by post-transcriptional mechanisms that probably contribute to acclimation of photosynthesis during extended periods of heat stress (DeRidder & Salvucci 2007)

High temperatures also require a high degree of saturation of the membrane lipids of the thylakoid for integrated functioning of its components and prevention of leakiness (Sharkey 2005) Therefore, not only Rubisco activity, but also membrane-bound processes of electron transport may be limiting photosynthesis at high temperatures

7.2 Effects of Low Temperatures on Photosynthesis

When plants grown at a moderate temperature are transferred to a lower temperature, but within the range normal for the growing season, photosynth-esis is initially reduced (Fig 34) Photon absorption is not affected by temperature, but the rate of elec-tron transport and biochemical processes are reduced as a direct consequence of the lower tem-perature Particularly, sucrose metabolism and/or phloem loading can become limiting for photo-synthesis, causing feedback inhibition (Fig 27) Acclimation to the lower growth temperature involves up-regulation of the limiting components of the photosynthetic apparatus Hence, the capacity

FIGURE35 Temperature dependence of Vmaxand the Km

of (A) the oxygenating and (B) the carboxylating reac-tion of Rubisco Vmaxis the rate of the carboxylating or

oxygenating reaction at a saturating concentration of CO2and O2, respectively The Kmis the concentration

of CO2and O2at which the carboxylating and

oxyge-nating reaction, respectively, proceed at the rate which equals 1/2Vmax Note that a logarithmic scale is used for

the y-axis and that the inverse of the absolute tempera-ture is plotted on the x-axis (‘‘Arrhenius-plot’’) In such a graph, the slope gives the activation energy, a measure for the temperature dependence of the reaction (Berry

& Raison 1981) (C) The combined effects of tempera-ture on kinetic properties as shown in (A) and (B) and relative solubility of O2and CO2(O/C) have been

mod-eled, normalized to values at 208C (D) Relative rates of the oxygenation and carboxylation reactions of Rubisco (Vo/Vc) and quantum yield (f CO2) modeled using the

same parameter values as in (C) For calculation of Vo/

Vcand f CO2, it was assumed that partial pressures of

CO2and O2in the chloroplast were 27 Pa and 21 kPa,

respectively Kinetic parameters used were calculated from Jordan and Ogren (1981) (courtesy I Terashima, The University of Tokyo, Japan)

for electron transport (Jmax) is increased, and Rubisco levels increase as well with the proportional increase in carboxylation capacity (Vcmax) (Atkin et al 2006) Feedback inhibition is alleviated by increased expression of enzymes of the sucrose synthesis pathway (Stitt & Hurry 2002) Acclimation comprises therefore an increase in photosynthetic capacity which is associated with an increase in leaf thickness, whereas chlorophyll concentrations remains more or less similar, thus causing an increase in Amax per unit chlorophyll The change is accompanied by a decrease in antenna size of PS II Hence, acclimation to low temperature resembles to a considerable extent acclimation to high irradi-ance (Huner et al 1998) In Plantago major (common plantain) species, the result of acclimation is that, just as with respiration (Fig 17 in Chapter 2B on respiration), photosynthetic rates are virtually inde-pendent of growth temperature (Fig 35)

When cold is more extreme, damage is likely to occur Many (sub)tropical plants grow poorly or become damaged at temperatures between 10 and 208C Such damage is called ‘‘chilling injury’’ and differs from frost damage, which only occurs below 08C Part of the chilling injury is associated with the photosynthetic apparatus The following aspects play a role:

1 Decrease in membrane fluidity

2 Changes in the activity of membrane-associated enzymes and processes, such as the photosyn-thetic electron transport

3 Loss of activity of cold-sensitive enzymes

Chilling resistance probably involves reduced saturationof membrane fatty acids which increases membrane fluidity and so compensates for the effect of low temperature on membrane fluidity (Chapter 4B on effects of radiation and temperature)

Chilling often leads to photoinhibition and photooxidation, because the biophysical reactions of photosynthesis (photon capture and transfer of excitation energy) are far less affected by tempera-ture than the biochemical steps, including electron transport and activity of the Calvin cycle (Sect 3.3) The leaves of evergreen plants in cold climates typi-cally develop and expand during the warm spring and summer months, and are retained during the winter months when all growth ceases Upon expo-sure to low temperature and high irradiance, the conversion of the light-harvesting violaxanthin to the energy-quenching zeaxanthin (Sect 3.3.1) occurs within minutes In addition to this ubiqui-tous process of ‘‘flexible dissipation’’, several forms of ‘‘sustained dissipation’’ exist The sustained dis-sipation does not relax upon darkening of the leaves, but it is still DpH-dependent; it is flexible in the sense that, e.g., warming of leaves allows this state to be quickly reversed The difference in the underlying mechanism between flexible and sus-tained DpH-independent dissipation is not related to zeaxanthin, because this xanthophyll is involved in both types of thermal dissipation (Demmig-Adams & (Demmig-Adams 2006) Therefore, under lasting stress conditions and in some plant species, the flex-ible, DpH-independent engagement and disengage-ment of zeaxanthin in dissipation is replaced by a

FIGURE36 Photosynthetic response to temperature in

plants from contrasting temperature regimes Curves from left to right are for Neuropogon acromelanus, an Antarctic lichen, Ambrosia chamissonis, a cool coastal

highly effective, but less flexible continuous engage-ment of zeaxanthin in dissipation that does not require a DpH It is not yet known which factors other than zeaxanthin are involved in the DpH-dependent, less flexible, but potent form of dissipa-tion that is particularly pronounced in long-lived, slow-growing evergreen species (Table 6)

Hardening of Thuja plicata (western red cedar) seedlings (i.e., acclimation to low temperatures) is associated with some loss of chlorophyll and with increased levels of carotenoids, giving the leaves a red-brown color Exposure to low temperatures causes a decline in photosynthetic capacity and the quantum yield of photosynthesis, as evi-denced by the decline in chlorophyll fluorescence (i.e., in the ratio Fv/Fm; Box 2A.4) The carotenoids prevent damage that might otherwise occur as a result of photooxidation (Sect 3.3.1) Upon trans-fer of the seedlings to a normal temperature (dehardening) the carotenoids disappear within a few days (Weger et al 1993) Other temperate con-ifers such as Pinus banksiana (jack pine) exhibit ‘‘purpling’’, which is caused by the accumulation of anthocyanin in epidermal cells This appears to protect the needles against photoinhibition of PS II through a simple screening of irradiance (Huner et al 1998) Accumulation of photoprotective antho-cyanins gives rise to typical autumn colors, e.g., in Cornus stolonifera(red-osier dogwood) (Feild et al 2001)

In the alpine and arctic species Oxyria digyna (alpine mountainsorrel), an increased resistance to photoinhibition is caused by an increased capacity to repair damaged PS II reaction centers and increased nonphotochemical quenching Maximum rates of photosynthesis by arctic and alpine plants measured in the field are similar to those of tempe-rate-zone species, but are reached at lower

temperatures—often 10—158C (Fig 36) These sub-stantial photosynthetic rates at low temperatures are achieved in part by high concentrations of Rubisco, as found in acclimation of lowland plants This may account for the high tissue N concentra-tion of arctic and alpine plants despite low N avail-ability in soils (K ¨orner & Larcher 1988) Although temperature optima of arctic and alpine plants are 10—308C lower than those of temperate plants, they are still 5—108C higher than average summer leaf temperatures in the field

8 Effects of Air Pollutants on Photosynthesis

Many air pollutants reduce plant growth, partly through their negative effects on photosynthesis Pollutants like SO2 and ozone (O3) that enter the leaf through stomata, directly damage the photo-synthetic cells of the leaf In general, any factor that increases stomatal conductance (e.g., high supply of water, high light intensity, high N supply) increases the movement of pollutants into the plant, and therefore their impact on photosynthesis At low [O3], decreased production Glycine max (soybean) corresponds to a decrease in leaf photosynthesis, but at higher [O3] the larger loss in production is associated with decreases in both leaf photosynth-esis and leaf area (Morgan et al 2003) Rates of net photosynthesis and stomatal conductance in Fagus sylvatica(beech) are about 25% lower when the O3 concentration is double that of the background con-centration in Kranzberg Forest (Germany), while Vcmax is and gm are not affected (Warren et al 2007) The major effect of SO2on growth and yield of Vicia faba (faba bean) is due to leaf injury (necrosis TABLE6 Differences in the response of photosynthesis and photoprotection between crops/weeds and ever-greens Typical changes in intrinsic photosynthetic capacity, DpH-independent dissipation, zeaxanthin and antheraxanthin (Z ỵ A) retention, in annual crops/biennial weeds vs evergreen species in response to transfer of shade-acclimated plants to high light or in response to the transition from summer to winter conditions

Shade to sun transfer Summer to winter transition

Annual crop Evergreen

Tropical annual/biennial

Crop/weed Temperate evergreen

Photosynthetic capacity " # " # #

DpH-independent dissipation * " " * " "

ZỵA retention * " " * " "

Source: Demmig-Adams & Adams (2006)

*Seen only transiently and at moderate levels upon transition.

and abscission of leaves), rather than direct effects on gas exchange characteristics (photosynthesis and respiration) (Kropf 1989)

9 C4Plants

9.1 Introduction

The first sections of this chapter dealt primarily with the characteristics of photosynthesis of C3 species There are also species with photosynthetic character-istics quite different from these C3plants These so-called C4 species belong to widely different taxo-nomic groups (Table 7); the C4 syndrome is very rare among tree species; Chamaesyce olowaluana (Euphorbiaceae) is a canopy-forming C4 tree from Hawaii (Sage 2004) Although their different anat-omy has been well documented for over a century, the biochemistry and physiology of C4 species has been elucidated more recently It is hard to say who first ‘‘discovered’’ the C4pathway of photosynthesis

(Hatch & Slack 1998); however, Hatch & Slack (1966) certainly deserve credit for combining earlier pieces of information with their own findings and propos-ing the basic pathway as outlined in this section

None of the metabolic reactions or anatomical features of C4plants are really unique to these spe-cies; however, they are all linked in a manner quite different from that in C3species Based on differ-ences in biochemistry, physiology, and anatomy, three subtypes of C4 species are discerned (Table 8) In addition, there are intermediate forms between C3and C4metabolism (Sect 9.6)

9.2 Biochemical and Anatomical Aspects

The anatomy of C4plants differs strikingly from that of C3plants (Fig 37) C4plants are characterized by their Kranz anatomy, a sheath of thick-walled cells surrounding the vascular bundle (‘‘Kranz’’ is the Ger-man word for ‘‘wreath’’) These thick walls of the bundle sheath cells may be impregnated with sub-erin, but this does not appear to be essential to reduce

TABLE The 19 families containing members with the C4 photo-synthetic pathway.*

Family

Number

of lineages Subtypes

Monocots

Poaceae 11 NADP-ME, NAD-ME, PCK Cyperaceae NADP-ME, NAD-ME Hydrocharitaceae Single-cell NADP-ME Dicots

Acanthaceae –

Aizoaceae NADP-ME

Amaranthaceae NADP-ME, NAD-ME Asteraceae NADP-ME

Boraginaceae NAD-ME Brassicaceae – Caryophyllaceae NAD-ME

Chenopodiaceae 10 NADP-ME, NAD-ME & single-cell NAD-ME

Euphorbiaceae NADP-ME Gisekiaceae NAD-ME Molluginaceae NAD-ME Nyctaginaceae NAD-ME Polygonaceae –

Portulacaceae NADP-ME, NAD-ME Scrophulariaceae –

Zygophyllaceae NADP-ME

Source: Sage (2004)

*

The number of lineages represents the putative times of independent evolu-tion of C4in the family The biochemical subtypes (not known for all species)

the gas diffusion between the bundle sheath and the mesophyll In some C4 species (NADP-ME types), the cells of the bundle sheath contain large chloro-plasts with mainly stroma thylakoids and very little grana The bundle sheath cells are connected via plasmodesmatawith the adjacent thin-walled meso-phyll cells, with large intercellular spaces

CO2 is first assimilated in the mesophyll cells, catalyzed by PEP carboxylase, a light-activated enzyme, located in the cytosol PEP carboxylase uses phosphoenolpyruvate (PEP) and HCO3 as substrates HCO3 is formed by hydratation of CO2, catalyzed by carbonic anhydrase The high affinity of PEP carboxylase for HCO3reduces Ci to about 100 mmol mol—1, less than half the C

iof C3 plants (Sect 2.2.2) PEP is produced in the light from pyruvate and ATP, catalyzed by pyruvate Pi -diki-nase, a light-activated enzyme located in the

chloroplast The product of the reaction catalyzed by PEP carboxylase is oxaloacetate, which is reduced to malate Alternatively, oxaloacetate may be transaminated in a reaction with alanine, forming aspartate Whether malate or aspartate, or a mixture of the two, are formed, depends on the subtype of the C4species (Table 8) Malate (or aspartate) dif-fuses via plasmodesmata to the vascular bundle sheath cells, where it is decarboxylated, producing CO2and pyruvate (or alanine) CO2is then fixed by Rubisco in the chloroplasts of the bundle sheath cells, which have a normal Calvin cycle, as in C3 plants Rubisco is not present in the mesophyll cells, which not have a complete Calvin cycle and only store starch when the bundle sheath chloroplasts reach their maximum starch concentrations

Fixation of CO2by PEP carboxylase and the sub-sequent decarboxylation occur relatively fast, TABLE8 Main differences between the three subtypes of C4species.*

Major substrate moving from

Subtype

Major decarboxylase in BSC

Decarboxylation

occurs in MC to BSC BSC to MC

Photosystems in BSC

NADP-ME NADP-malic enzyme Chloroplast Malate Pyruvate I and IIa

NAD-ME NAD-malic enzyme Mitochondria Aspartate Alanine I and II PCK PEP carboxykinase Cytosol Aspartate ỵ malate Alanine ỵ PEP I and II

*MC is mesophyll cells; BSC is vascular bundle sheath cells.

aSome NADP-ME monocots, including Zea mays (corn) have only PS I in BSC chloroplasts.

FIGURE 37 (Facing page) Schematic representation

photosynthetic metabolism in the three C4types

distin-guished according to the decarboxylating enzyme NADP-ME, NADP-requiring malic enzyme; PCK, PEP carboxykinase; NAD-ME, NAD-requiring malic enzyme Numbers refer to enzymes (1) PEP carboxy-lase, (2) NADP-malate dehydrogenase, (3) NADP-malic enzyme, (4) pyruvate Pi-dikinase, (5) Rubisco, (6) PEP

carboxykinase, (7) alanine aminotransferase, (8) aspar-tate amino transferase, (9) NAD-malate dehydrogenase, (10) NAD-malic enzyme (after Lawlor 1993) (Above) Cross-sections of leaves of monocotyledonous C4

grasses (Ghannoum et al 2005) Chlorophyll a

autofluorescence of a leaf cross-section of (Left) Pani-cum miliaceum (French millet, NAD-ME), and (Right) Sorgum bicolor (millet, NAD-ME) The images were obtained using confocal microscopy Cell walls are shown in green and chlorophyll a autofluorescence in red Most of the autofluorescence emanates from bun-dle sheath cells in the NAD-ME species (Left) and from the mesophyll cells in the NADP-ME species (Right), showing the difference in chlorophyll distribution between the two subtypes (courtesy O Ghannoum, Centre for Horticulture and Plant Sciences, University of Western Sydney, Australia) Copyright American Society of Plant Biologists

allowing the build-up of a high concentration of CO2 in the vascular bundle sheath When the outside CO2 concentration is 380 mmol mol—1, that at the site of Rubisco in the chloroplasts of the vascular bundle is 1000—2000 mmol mol—1 The Ci, that is the CO2concentration in the intercellular spaces in the mesophyll, is only about 100 mmol mol—1 With such a steep gradient in the CO2concentration it is inevi-table that some CO2diffuses back from the bundle sheath to the mesophyll, but this is only about 20% In other words, C4 plants have a mechanism to enhance the CO2concentration at the site of Rubisco to an extent that its oxygenation reaction is virtually fully inhibited Consequently, C4plants have negli-gible rates of photorespiration

Based on the enzyme involved in the decarbox-ylation of the C4compounds transported to the vas-cular bundle sheath, three groups of C4species are discerned: NADP-malic enzyme-, NAD-malic enzyme- and PEP carboxykinase-types (Table 8, Fig 37) The difference in biochemistry is closely correlated with anatomical features of the bundle sheath and mesophyll of the leaf blade as viewed in transverse sections with the light microscope (Ellis 1977) In NAD-ME-subtypes, which decarbox-ylate malate (produced from imported aspartate) in the bundle sheath mitochondria, the mitochondrial frequency is several-fold higher than that in NADP-ME-subtypes The specific activity of the mitochon-drial enzymes involved in C4photosynthesis is also greatly enhanced (Hatch & Carnal 1992) The NAD-ME group of C4species tends to occupy the driest habitats, although the reason for this is unclear (Ellis et al 1980, Ehleringer & Monson 1993)

Decarboxylation of malate occurs only during assimilation of CO2, and vice versa The explanation for this is that the NADP needed to decarboxylate malate is produced in the Calvin cycle, during the assimilation of CO2 At least in the more ‘‘sophisti-cated’’ NADP-ME C4plants such as Zea mays (corn) and Saccharum officinale (sugar cane), the NADPH required for the photosynthetic reduction of CO2 originates from the activity of NADP malic enzyme Since two molecules of NADPH are required per molecule of CO2fixed by Rubisco, this amount of NADPH is not sufficient for the assimilation of all CO2 Additional NADPH is required to an even larger extent if aspartate, or a combination of malate and aspartate, diffuses to the bundle sheath It is assumed that this additional NADPH can be imported via a ‘‘shuttle’’, involving PGA and dihy-droxyacetone phosphate (DHAP) Part of the PGA that originates in the bundle-sheath chloroplasts returns to the mesophyll Here it is reduced, produ-cing DHAP, which diffuses to the bundle sheath

Alternatively, NADPH required in the bundle sheath cells might originate from the removal of electrons from water This reaction requires the activity of PS II, next to PS I PS II is only poorly developed in the bundle sheath cells, at least in the ‘‘more sophisticated’’ C4species The poor develop-ment of PS II activity in the bundle sheath indicates that very little O2is evolved in these cells that con-tain Rubisco, which greatly favors the carboxylation reaction over the oxygenation

The formation of PEP from pyruvate in the meso-phyll cells catalyzed by pyruvate Pi-dikinase, requires one molecule of ATP and produces AMP, instead of ADP; this corresponds to the equivalent of two molecules of ATP per molecule of PEP This represents the metabolic costs of the CO2pumpof the C4pathway It reduces photosynthetic efficiency of C4plants, when compared with that of C3plants under nonphotorespiratory conditions In sum-mary, C4 photosynthesis concentrates CO2 at the site of carboxylation by Rubisco in the bundle sheath, but this is accomplished at a metabolic cost

9.3 Intercellular and Intracellular

Transport of Metabolites of the C4Pathway

Transport of the metabolites that move between the two cell types occurs by diffusion through plasmo-desmata The concentration gradient between the mesophyll and bundle sheath cells is sufficiently high to allow diffusion at a rate that readily sustains photosynthesis, with the exception of that of pyru-vate How can we account for rapid transport of pyruvate from the bundle sheath to the mesophyll if there is no concentration gradient?

Uptake of pyruvate in the chloroplasts of the mesophyll cells is a light-dependent process, requir-ing a specific energy-dependent carrier Active uptake of pyruvate into the chloroplast reduces the pyruvate concentration in the cytosol of these meso-phyll cells to a low level, creating a concentration gradient that drives diffusion from the bundle sheath cells (Fluăgge et al 1985)

In the chloroplasts of the mesophyll cells, pyru-vate is converted into PEP, which is exported to the cytosol in exchange for Pi The same translocator that facilitates this transport is probably also used to export triose-phosphate in exchange for PGA This translocator operates in the reverse direction in mesophyll and bundle sheath chloroplasts, in that PGA is imported and triose-phosphate is exported in the mesophyll chloroplasts, while the chloro-plasts in the bundle sheath export PGA and import triose phosphate

The chloroplast envelope of the mesophyll cells also contains a translocator for the transport of dicar-boxylates (malate, oxaloacetate, aspartate, and gluta-mate) Transport of these carboxylates occurs by exchange The uptake of oxaloacetate, in exchange for other dicarboxylates, is competitively inhibited by these other dicarboxylates, with the values for Ki being in the same range as those for Km [Kiis the inhibitor (i.e., dicarboxylate) concentration at which the inhibition of the transport process is half that of the maximum inhibition by that inhibitor; Kmis the substrate (oxaloacetate) concentration at which the transport process occurs at half the maximum rate.] Such a system does not allow rapid import of oxaloa-cetate A special transport system, transporting oxa-loacetate without exchange against other dicarboxylates, takes care of rapid import of oxaloa-cetate into the mesophyll chloroplasts

9.4 Photosynthetic Efficiency and Performance at High and Low Temperatures

The differences in anatomy and biochemistry result in strikingly different An-Cicurves between C3and

C4 First, the CO2-compensation pointof C4plants is only 0—5 mmol mol—1 CO

2, as compared with 40—50 mmol mol—1 in C3 plants (Fig 38) Second, this compensation point is not affected by O2 con-centration, as opposed to that of C3plants which is considerably less at a low O2 concentration (i.e., when photorespiration is suppressed) Thirdly, the Ci(the internal concentration of CO2in the meso-phyll) at a Ca of 380 mmol mol—1 is only about 100 mmol mol—1, compared with approximately 250 mmol mol—1in C

3plants (Fig 38)

There are also major differences in the character-istics of the light-response curves of CO2 assimila-tion of C3and C4 species The initial slope of the light-response represents the light-limited part and is referred to as the quantum yield Photochemical activity is limited by the rate of electron transport under these conditions Changes in quantum yield are thus caused by changes in the partitioning between carboxylation and oxygenation reactions of Rubisco When measured at 308C or higher, the quantum yield is considerably higher for C4plants and independent of the O2concentration, in contrast to that of C3 plants Therefore, at relatively high temperatures, the quantum yield of photosynthesis

FIGURE38 Response of net photosynthesis (An) to

inter-cellular CO2concentration in the mesophyll (Ci) of C3

and C4plants C3plants respond strongly to O2as shown

by the lines for normal atmospheric (21%) and low (2%) O2concentrations, whereas C4plants not The CO2

-response curves were calculated based on models

described by Von Caemmerer (2000) Parameter values for the C3model were Vcmax¼150 and Jmax¼225 mmol

m–2s–1(see Box 2A.1), and in the C4model Vcmax¼60

and Vpmax¼120 mmol m–2s–1, where Vpmaxis the

is higher for C4plants, and is not affected by tem-perature By contrast, the quantum yield of C3 plants declines with increasing temperature, due to the proportionally increasing oxygenating activity of Rubisco (Fig 39) At an atmospheric O2and CO2 concentration of 21% and 350 mmol mol—1, respec-tively, the quantum yield is higher for C4plants at high temperatures due to photorespiration in C3 species, but lower at low temperatures due to the additional ATP required to regenerate PEP in C4 species When measured at a low O2concentration

(to suppress photorespiration) and a Caof 350 mmol mol—1, the quantum yield is invariably higher for C

3 plants

The rate of CO2assimilation of C4plants typi-cally saturates at higher irradiance than that of C3 plants, because Amaxof C4plants is generally higher This is facilitated by a high Cc, the CO2 concentra-tion at the site of Rubisco In C3 plants with their generally lower Amax, the light-response curve levels off at lower irradiances, because CO2becomes the limiting factor for the net CO2assimilation At

TABLE Variation in kinetic parameters of the ubiquitous carboxylating enzyme Rubisco at 258C for eight species in four groups.*

Km(CO2)

Presence In water In air

of CCM mM mmol mol1 kcatS1

Cyanobacteria

Synechococcus ỵ 293 12.5

Green algae

Chlamydomonas reinhardtii ỵ 29 5.8 C4terrestrial plants

Amaranthus hybridus ỵ 16 480 3.8

Sorghum bicolor ỵ 30 900 5.4

Zea mays ỵ 34 1020 4.4

C3terrestrial plants

Triticum aestivum – 14 420 2.5

Spinacia oleracea – 14 420 3.7

Nicotiana tabacum – 11 330 3.4

Source: Tcherkez et al (2006)

*

Shown are the Michaelis–Menten constant Km(CO2), inversely related to substrate

(CO2) affinity, and the catalytic turnover rate at saturating CO2(kcat, mol CO2(mol

catalytic sites)–1 Km(CO2) in air is calculated from the value provided for water using

the solubility of CO2at 258C (33.5 mmol L–1at standard atmospheric pressure) CCM ¼

carbon-concentrating mechanism

FIGURE39 The effect of temperature and the intercellular CO2concentration (Ci) on the quantum yield of the

photosynthetic CO2assimilation in a C3and a C4plant (after Ehleringer & Bj ăorkman 1977)

increasing atmospheric CO2concentrations the irra-diance at which light saturation is reached shifts to higher levels also in C3plants

The high concentration of CO2in the vascular bundle sheath of C4-plants, the site of Rubisco, allows different kinetic properties of Rubisco Table shows that indeed the Km(CO2) of Rubisco from terrestrial C3plants is lower than that from C4 plants A high Km, that is a low affinity, for CO2of Rubisco is not a disadvantage for the photosynthesis of C4plants, considering the high Ccin de the bun-dle sheath For C3plants a low Kmfor CO2is vital, since the Ciis far from saturating for Rubisco in their mesophyll cells The advantage of the high Kmof the C4Rubisco is thought to be indirect in that it allows a high maximum rate per unit protein of the enzyme (Vmaxor kcat) That is, the tighter CO2is bound to Rubisco, the longer it takes for the carboxylation to be completed In C3plants, a high affinity is essen-tial, so that kcatcannot be high C4plants, which not require a high affinity, indeed have an enzyme with a high kcat, allowing more moles of CO2 to be fixed per unit Rubisco and time at the high Cc(Table 10) Interestingly, the alga Chlamydo-monas reinhardtii, which has a CO2-concentrating mechanism (Sect 11.3), also has a Rubisco enzyme with a high Km (low affinity) for CO2and a high Vmaxand kcat(Table 10) Apparently, there is a

trade-off in Rubisco between CO2 specificity [a low Km(CO2)] and catalytic capacity (a high kcat)

The biochemical and physiological differences between C4and C3plants have important ecologi-cal implications The abundance of C4monocots in regional floras correlates most strongly with grow-ing season temperature, whereas C4 dicot abun-dance correlates more strongly with aridity and salinity (Ehleringer & Monson 1993) At regional and local scales, areas with warm-season rainfall have greater C4abundance than regions with cool-season precipitation Along local gradients, C4 spe-cies occupy microsites that are warmest or have driest soils In communities with both C3 and C4 species, C3 species are most active early in the growing season when conditions are cool and moist, whereas C4activity increases as conditions become warmer and drier Together these patterns suggest that high photosynthetic rates at high tem-perature (due to lack of photorespiration) and high water-use efficiency (WUE) (due to the low Ci, which enables C4plants to have a lower stoma-tal conductancefor the same CO2assimilation rate) are the major factors governing the ecological dis-tribution of the C4 photosynthetic pathway Any competitive advantage of the high WUE of C4 plants, however, has been difficult to document experimentally (Ehleringer & Monson 1993) This TABLE 10 The number of chloroplasts and of mitochondria plus peroxisomes

in bundle sheath cells compared with those in mesophyll cells (BSC/MC) and the CO2-compensation point (, mmol mol–1, of C3, C4, and C3–C4intermediates belong-ing to the genera Panicum, Neurachne, Flaveria, and Moricandia

BSC/MC

Species

Photosynthetic

pathway Chloroplasts

Mitochondria ỵ peroxisomes 

P milioides C3C4 0.9 2.4 19

P miliaceum C4 1.1 8.4

N minor C3–C4 3.1 20.0

N munroi C4 0.8 4.9

N tenuifolia C3 0.6 1.2 43

F anomala C3–C4 0.9 2.3

F floridana C3–C4 1.4 5.0

F linearis C3–C4 2.0 3.6 12

F oppositifolia C3–C4 1.4 3.6 14

F brownii C4-like 4.2 7.9

F trinerva C4 2.2 2.4

F pringlei C3 0.5 1.0 43

M arvensis C3–C4 1.4 5.2 32

M spinosa C3–C4 1.6 6.0 25

M foleyi C3 1.5 3.3 51

M moricandioides C3 2.0 2.8 52

may well be due to the fact that, in a competitive situation, any water that is left in the soil by a plant with a high WUE is available for a competitor with lower WUE Although WUE of C4plants is higher, the C4 pathway does not give them a higher drought tolerance

C4plants generally have lower tissue N concen-trations, because they have 3—6 times less Rubisco than C4plants and very low levels of the photore-spiratory enzymes, though some of the advantage is lost by the investment of N in the enzymes of the C4 pathway C4plants also have equivalent or higher photosynthetic rates than C3plants, resulting in a higher rate of photosynthesis per unit of leaf N (Photosynthetic N-Use Efficiency, PNUE), espe-cially at high temperatures (Fig 40) The higher PNUE of C4plants is accounted for by: (1) suppres-sion of the oxygenase activity of Rubisco, so that the enzyme is only used for the carboxylation reaction; (2) the lack of photorespiratory enzymes; (3) the higher catalytic activity of Rubisco due to its high kcatand the high Cc(Table 10) Just as in a compar-ison of C3species that differ in PNUE (Sect 4.2.1 of Chapter on mineral nutrition), there is no consis-tent tendency of C4species to have increased abun-dance or a competitive advantage in low-N soils (Christie & Detling 1982, Sage & Pearcy 1987a) This suggests that the high PNUE of C4species is less important than their high WUE and high opti-mum temperature of photosynthesis in explaining patterns of distribution

One of the key enzymes of the C4pathway in Zea mays (corn), pyruvate Pi-dikinase, readily loses its activity at low temperature and hence the leaves’ photosynthetic capacity declines This accounts for part of the chilling sensitivity of most C4plants Loss

of activity of pyruvate Pi-dikinase at low tempera-tures can be prevented by protective (‘‘compatible’’) compounds (Sect 3.4.5 of Chapter on plant water relations), but it remains to be investigated if this plays a major role in intact C4plants (Krall et al 1989)

9.5 C3–C4Intermediates

In the beginning of the 1970s, when the C4pathway was unraveled, there were attempts to cross C3and C4species of Atriplex (saltbush) This was considered a useful approach to enhance the rate or efficiency of photosynthesis and yield of C3parents The complex-ity of anatomy and biochemistry of the C4plants, however, is such, that these crosses have not pro-duced any useful progeny (Brown & Bouton 1993) Since molecular techniques have become available which allow silencing and over-expression of specific genes in specific cells, attempts have been made to reduce the activity of glycine decarboxylase, the key enzyme in photorespiration, in mesophyll cells of C3 plants and over-express the gene in the bundle sheath Although these attempts have been success-ful from a molecular point of view in that the aim of selectively modifying the enzyme activity was achieved, no results have yet been obtained to show enhanced rates of photosynthesis This is perhaps not unexpected, in view of the rather small advantage true C3—C4intermediates are likely to have in com-parison with C3relatives

Further attempts to transform C3 crops into C4 were inspired by the discovery of plants that perform a C4pathway without intercellular compartmenta-tion between mesophyll and bundlesheath Suaeda aralocaspica(formerly known as Borszczowia

aralocas-FIGURE40 The rate of CO2assimilation as a function of

the organic N concentration in the leaf and the tempera-ture, as measured for the C3plant Chenopodium album

(pigweed, circles) and the C4plant Amaranthus

retro-flexus (triangles) (after Sage & Pearcy 1987b) Copyright American Society of Plant Biologists

pica, seepweed) and Bienertia cycloptera have the com-plete C4cycle operating in mesophyll cells PEP car-boxylation and regeneration occur at the distal ends of the cell exposed to the intercellular air spaces The C4acids produced must therefore diffuse from here to the opposite, proximal end of the cell where they are decarboxylated An elongated vacuole provides high resistance to CO2efflux and thus CO2 accumu-lates where Rubisco is located In this regard, the general layout of these C3—C4intermediates is similar to that of Kranz-type C4plants, the major difference being the lack of a cell wall segregating the PCA and PCR compartments (Sage 2002, 2004) The existence of single-cell C4in terrestrial plants opens new pos-sibilities for introducing the C4pathway in C3crops, because it does not require complicated anatomical changes (Surridge 2002)

Over 20 plant species exhibit photosynthetic traits that are intermediate between C3and C4plants (e.g., species in the genera Alternanthera, Flaveria, Neurachne, Moricandia, Panicum, and Parthenium) These show reduced rates ofphotorespiration and CO2-compensation points in the range of to 35 mmol mol—1, compared with 40—50 mmol mol—1in C3and to mmol mol—1in C4plants (Table 10) They have a weakly developed Kranz anatomy, com-pared with the true C4 species, but Rubisco is located both in the mesophyll and the bundle sheath cells (Brown & Bouton 1993)

Two main types of intermediates are distin-guished In the first type (e.g., Alternanthera ficoides, Alternanthera enella, Moricandia arvensis, and Pani-cum milioides) the activity of key enzymes of the C4 pathway is very low, and they not have a func-tional C4 acid cycle Their low CO2-compensation point is due to the light-dependent recapture by mesophyll cells of CO2released in photorespiration in the bundle sheath cells, which contain a large fraction of the organelles involved in photorespira-tion, compared with that in C3species (Table 10) In these C3—C4intermediates a system has evolved to salvage CO2escaping from the bundle sheath cells, but they not have the CO2-concentrating mechanism of true C4species (Ehleringer & Monson 1993) In the leaves of this type of intermediate spe-cies, glycine decarboxylase, a key enzyme in photo-respiration that releases the photorespiratory CO2, occurs exclusively in the cells surrounding the vas-cular bundle sheath (Morgan et al 1992) Products of the oxygenation reaction, including glycine, prob-ably move to the bundle sheath cells Presumprob-ably, the products are metabolized in the bundle sheath, so that serine can move back to the mesophyll (Fig 41) Due to the exclusive location of glycine decarboxylase in the bundle sheath cells, the release of CO2in photorespiration occurs close to the vas-cular tissue, with chloroplasts occurring between these mitochondria and the intercellular spaces

FIGURE41 A model of the photorespiratory metabolism

in leaves of the C3–C4intermediate Moricandia arvensis,

showing the recapture of CO2released by glycine

dec-arboxylase The model accounts for the low

CO2-compensation point and the low apparent rate of

Glycine decarboxylase is only found in the enlarged mitochondria arranged along the cell walls adjacent to the vascular tissue and overlain by chloroplasts This location of glycine decarboxylase increases the diffusion path for CO2between the site of release and the atmosphere and allows the recapture of a large fraction of the photorespiratory CO2, released by glycine decarboxylase, by Rubisco located in the bundle sheath The location of glycine decarboxy-lase in the bundle sheath allows some build-up of CO2, but not to the same extent as in the true C4 plants Since the oxygenation reaction of Rubisco is only suppressed in the bundle sheath, and there probably only partly, whereas oxygenation in the mesophyll cells occurs to the same extent as in C3 plants, the advantage in terms of the net rate of CO2 assimilation is rather small, compared with that in a true C4plant (Von Caemmerer 1989)

In the second type of intermediate species (e.g., Flaveria anomalaand Neurachne minor), the activity of key enzymes of the C4 pathway is considerable Rapid fixation of14CO2into C4acids, followed by transfer of the label to Calvin-cycle intermediates, has been demonstrated These species have a lim-ited capacity for C4photosynthesis, but lower quan-tum yields than either C3or C4, presumably because the operation of the C4cycle in these plants does not really lead to a concentration of CO2to the extent it does in true C4species

In addition to the C3—C4 intermediate species, there are some species [e.g., Eleocharis vivipara (sprouting spikerush) and Nicotiana tabacum (tobacco)] that are capable of either C3or C4 photo-synthesis in different tissues (Ueno et al 1988, Ueno 2001) Tobacco, a typical C3plant, shows character-istics of C4 photosynthesis in cells of stems and petioles that surround the xylem and phloem; these cells are supplied with carbon for photosynth-esis from the vascular system, and not from stomata These photosynthetic cells possess high activities of enzymes characteristic of C4photosynthesis which allows the decarboxylation of four-carbon organic acids from the xylem and phloem, thus releasing CO2for photosynthesis (Hibberd & Quick 2002)

C4plants that can shift to a CAM mode occur in the genus Portulaca (Sect 10.4)

9.6 Evolution and Distribution of C4Species

C4species represent approximately 5% of all higher plant species, C3species accounting for about 85% and CAM species (Sect 10) for 10% C4 photosynth-esis first arose in grasses, 24—35 million years ago,

and in dicots 15—21 million years ago (Sage 2004) However, it took several millions of years before the C4 pathway spread on several continents and became dominant over large areas, between and million years ago, as indicated by changes in the carbon-isotope ratios of fossil tooth enamel in Asia, Africa, North America, and South America (Cerling et al 1997) A decreasing atmospheric CO2 concen-tration, as a result of the photosynthetic activity of plants and possibly much more so due to tectonic and subsequent geochemical events, has been a sig-nificant factor contributing to C4evolution Briefly, the collision of the Indian subcontinent caused the uplift of the Tibetan Plateau With this, Earth crust consuming CO2became exposed over a vast area The reaction CaSiO3ỵ CO2< -> CaCO3ỵ SiO2is responsible for the dramatic decline in atmospheric CO2 concentration (Raymo & Ruddiman 1992, Ehleringer & Monson 1993) Since CO2levels were already low when the first C4plants evolved, other factors must have been responsible for the rapid spread of C4 plants many millions of years after they first arose

The universal carboxylating enzyme Rubisco does not operate efficiently at the present low CO2 and high O2 atmospheric conditions Low atmo-spheric CO2concentrations would increase photo-respiration and thus favor the CO2-concentrating mechanisms and lack of photorespiration that characterize C4species Considering the three sub-types of C4species and their occurrence in at least 19 different families of widely different taxonomic groups, C4plants must have evolved from C3 ances-tors independently about 48 times on different con-tinents (convergent evolution) (Table 7) Morphological and eco-geographical information combined with molecular evidence suggests that C4 photosynthesis has evolved twice in different lineages within the genus Flaveria (Sage 2004) The physiology of C3—C4intermediates suggests that the mechanism to recapture CO2 evolved before the CO2-concentrating mechanism (Sect 9.5) The phy-logeny of Flaveria species, as deduced from an ana-lysis of the nucleotide sequences encoding a subunit of glycine decarboxylase, suggests that C4species originated from C3—C4intermediates, and that C4in this genus developed relatively recently (Sage 2004) C4photosynthesis originated in arid regions of low latitude, where high temperatures in combina-tion with drought and/or salinity, due to a globally drying climate and increased fire frequency, pro-moted the spread of C4 plants (Keeley & Rundel 2005, Beerling & Osborne 2006) A major role for climatic factors as the driving force for C4evolution is also indicated by C4 distributions in

Mesoamerican sites that have experienced contrast-ing moisture variations since the last glacial max-imum Analyses of the carbon-isotope composition of leaf wax components indicate that regional cli-mate exerts a strong control over the relative abun-dance of C3and C4species, and that in the absence of favorable moisture and temperature conditions a low atmospheric CO2concentration alone does not favor C4expansion (Huang et al 2001)

Low altitudes in tropical areas continue to be centers of distribution of C4species Tropical and temperate lowland grasslands, with abundant warm-season precipitation, are dominated by C4 species At higher elevations in these regions C3 species are dominant, both in cover and in composi-tion, for example on the summits of the Drakenberg in South Africa (Vogel et al 1978) and on highland

plains in a temperate arid region of Argentinia (Cavagnaro 1988)

The high concentration of CO2 at the site of Rubisco, allows net CO2assimilation at relatively high temperatures, where photorespiration results in low net photosynthesis of C3species due to the increased oxygenating activity of Rubisco This explains why C4 species naturally occur in warm, open ecosystems, where C3species are less success-ful (Figs 42 and 43) There is no a priori reason, however, why C4photosynthesis could not function in cooler climates The lower quantum yield of C4 species at low temperature would be important in dense canopies where light limits photosynthesis (and where quantum yield is therefore important) Quantum yield, however, is less important at higher levels of irradiance, and there is quite a wide

tem-FIGURE42 Geographic

distri-bution of C4species in North

America Left: percentage of grass taxa that are C4plants

Right: percentage of dicotyle-don taxa that are C4plants in

regional floras of North America (Teeri & Stowe 1976, and Stowe & Teeri 1978, as cited in Osmond et al 1982)

FIGURE43 Left: The percentage occurrence of C4

meta-bolism in grass floras of Australia in relation to tempera-ture in the growing season (January) Right: The

percentage occurrence of C4grass species of the three

perature range where the quantum yield is still high compared with that of C3plants (Fig 39) The high sensitivity to low temperature of pyruvate Pi -dikinase, a key enzyme in the C4pathway may be the main reason why C4 species have rarely expanded to cooler places (Sect 7.2) Compatible solutes can decrease the low-temperature sensitivity of this enzyme and this could allow the expansion of C4species into more temperate regions in the future Alternatively, rising atmospheric CO2concentration may offset the advantages of the CO2-concentrating mechanism of C4photosynthesis (Sect 12)

9.7 Carbon-Isotope Composition of C4Species

Although Rubisco of C4 plants discriminates between12CO

2and13CO2, just like that of C3plants, the fractionation in C4species is considerably less than that in C3plants This is explained by the small extent to which inorganic carbon diffuses back from the vascular bundle to the mesophyll (Sect 9.2) Moreover, the inorganic carbon that does dif-fuse back to the mesophyll cells will be refixed by PEP carboxylase, which has a very high affinity for bicarbonate (Box 2A.2) Most of the13CO2that accu-mulates in the bundle sheath is ultimately assimi-lated; hence the isotope fractionation of CO2is very small in C4species (Fig 44)

The isotopic differences between C3 and C4 plants (Fig 44) are large compared with isotopic changes occurring during digestion by herbivores or decomposition by soil microbes This makes it possible to determine the relative abundance of C3 and C4species in the diets of animals by analyzing tissue samples of animals (‘‘You are what you eat’’) or as sources of soil organic matter in paleosols (old soils) These studies have shown that many

general-ist herbivores show a preference for C3rather than C4plants (Ehleringer & Monson 1993) C3species, however, also tend to have more toxic secondary metabolites, which cause other herbivores to show exactly the opposite preference

10 CAM Plants

10.1 Introduction

In addition to C3and C4 species, there are many succulent plants with another photosynthetic path-way: Crassulacean Acid Metabolism (CAM) This pathway is named after the Crassulaceae, a family in which many species show this type of metabolism CAM, however, also occurs commonly in other families, such as the Cactaceae, Euphorbiaceae, Orchidaceae, and Bromeliaceae [e.g., Ananas como-sus(pineapple)] There are about 10000 CAM spe-cies from 25 to 30 families (Table 11), all angiosperms, with the exception of a few fern spe-cies that also have CAM characteristics

The unusual capacity of CAM plants to fix CO2 into organic acids in the dark, causing nocturnal acidification, with de-acidification during the day, has been known for almost two centuries A full appreciation of CAM as a photosynthetic process was greatly stimulated by analogies with C4species The productivity of most CAM plants is fairly low This is not an inherent trait of CAM species, however, because some cultivated CAM plants (e.g., Agave mapisagaand Agave salmiana) may achieve an average above-ground productivity of kg dry mass m—2yr—1 An even higher productivity has been observed for irrigated, fertilized, and carefully pruned Opuntia amyclea and Opuntia ficus-indica

TABLE 11 Taxonomic survey of flowering plant families known to have species showing crassulacean acid metabolism (CAM) in different taxa

Agavaceae Geraniaceae Aizoaceae Gesneriaceae Asclepidiaceae Labiatae Asteraceae Liliaceae Bromeliaceae Oxalidaceae Cactaceae Orchidaceae Clusiaceae Piperaceae Crassulaceae Polypodiaceae Cucurbitaceae Portulacaceae Didieraceae Rubiaceae Euphorbiaceae Vitaceae

Source: Kluge & Ting (1978) and Medina (1996)

FIGURE44 The carbon-isotope composition of C3, C4,

and CAM plants (Sternberg et al 1984)

(prickly pears) (4.6 kg m—2yr—1; Nobel et al 1992) These are among the highest productivities reported for any species In a comparison of two succulent species with similar growth forms, Cotyledon orbicu-lata(pig’s ear) (CAM) and Othonna optima (C3), dur-ing the transition from the rainy season to subsequent drought, the daily net rate of CO2 assim-ilation is similar for the two species This shows that rates of photosynthesis of CAM plants may be as high as those of C3plants, if morphologically similar plants adapted to the similar habitats are compared (Eller & Ferrari 1997)

As with C4plants, none of the enzymes or meta-bolic reactions of CAM are really unique to these species The reactions proceed at different times of the day, however, quite distinct from C3 and C4 species Based on differences in the major decarbox-ylating enzyme, two subtypes of CAM species are discerned (Sect 10.2) In addition, there are inter-mediate forms between C3 and CAM, as well as facultative CAM plants (Sect 10.4)

10.2 Physiological, Biochemical, and Anatomical Aspects

CAM plants are characterized by their succulence (but this is not pronounced in epiphytic CAM plants; Sect 10.5), the capacity to fix CO2at night via PEP carboxylase, the accumulation of malic acid in the vacuole, and subsequent de-acidification dur-ing the day, when CO2is released from malic acid and fixed in the Calvin cycle, using Rubisco

CAM plants show a strong fluctuation in pH of the cell sap, due to the synthesis and breakdown of malic acid The concentration of this acid may increase to 100 mM By isolating vacuoles of the CAM plant Kalanchoe daigremontiana (devil’s back-bone), it was shown that at least 90% of all the acid in the cells is in the vacuole The kinetics of malic acid efflux from the leaves of Kalanchoe daigremontiana provides further evidence for the predominant loca-tion of malic acid in the vacuole

At night, CO2is fixed in the cytosol, catalyzed by PEP carboxylase, producing oxaloacetate (Fig 45) PEP originates from the breakdown of glucose in glycolysis; glucose is formed from starch Oxaloace-tate is immediately reduced to malate, catalyzed by malate dehydrogenase Malate is transported to the large vacuoles in an energy-dependent manner A H+-ATPase and a pyrophosphatase pump H+into the vacuole, so that malate can move down an elec-trochemical potential gradient (Sect 2.2.2 of Chapter

6 on mineral nutrition) In the vacuole it will be present as malic acid

The release of malic acid from the vacuole dur-ing the day is supposedly passive Upon release it is decarboxylated, catalyzed by malic enzyme (NAD- or NADP-dependent), or by PEP carboxy-kinase(PEPCK) Like C4species, CAM species are subdivided depending on the decarboxylating enzyme The malic enzyme subtypes (ME-CAM) have a cytosolic NADP-malic enzyme, as well as a mitochondrial NAD-malic enzyme; they use a chloroplastic pyruvate Pi-dikinase to convert the C3fragment originating from the decarboxylation reaction into carbohydrate via PEP PEPCK-type CAM plants have very low malic enzyme activities (as opposed to PEPCK-C4plants) and no pyruvate Pi-dikinase activity, but high activities of PEP carboxykinase

The C3 fragment (pyruvate or PEP) that is formed during the decarboxylation, is converted into starch and the CO2that is released is fixed by Rubisco, much the same as in C3 plants During the decarboxylation of malic acid and the fixation of CO2by Rubisco in the Calvin cycle, the stomata are closed They are open during the nocturnal fixation of CO2

The CAM traits can be summarized as follows:

1 Fluctuation of organic acids, mainly of malic acid, during a diurnal cycle;

2 Fluctuation of the concentration of sugars and starch, opposite to the fluctuation of malic acid; A high activity of PEP carboxylase (at night) and

of a decarboxylase (during the day);

4 Large vacuoles in cells containing chloroplasts; Some degree of succulence;

6 The CO2assimilation by the leaves occurs predo-minantly at night

under laboratory conditions, following an abrupt dark-to-light transition, but is not apparent under natural conditions In phase III the stomata are fully closed and malic acid is decarboxylated The Cimay then increase to values above 10000 mmol mol—1 This is when normal C3 photosynthesis takes place and when sugars and starch accumu-late When malic acid is depleted, the stomata open again, possibly because Cidrops to a low level; this is the beginning of phase IV Gradually more exo-genous and less endoexo-genous CO2is being fixed by Rubisco In this last phase, CO2may be fixed by PEP carboxylase again, as indicated by the

photosynthetic quotient (PQ), i.e., the ratio of O2 release and CO2uptake Over an entire day the PQ is about (Table 12), but deviations from this value occur, depending on the carboxylation process (Fig 47)

In phase III, when the stomata are fully closed, malic acid is decarboxylated, and the Ciis very high, photorespirationis suppressed, as indicated by the relatively slow rate of O2uptake (as measured using 18

O2; Fig 47) In phase IV, when malic acid is depleted and the stomata open again, photorespira-tion does occur, as demonstrated by increased uptake of18O2

FIGURE 45 Metabolic pathway and cellular

compart-mentation of Crassulacean Acid Metabolism (CAM), showing the separation in night and day of

carboxylation and decarboxylation The steps specific for PEPCK-CAM plants are depicted in red

How CAM plants regulate the activity of the two carboxylating enzymes and decarboxylating enzymes in a coordinated way to avoid futile cycles? Rubisco is inactive at night for the same reason as in C3plants: this enzyme is part of the Calvin cycle that depends on the light reactions and is inactivated in the dark (Sect 3.4.2) In addition, the kinetic proper-ties of PEP carboxylase are modulated In Mesem-bryanthemum crystallinum(ice plant) and in Crassula argentea(jade plant), PEP carboxylase occurs in two configurations: a ‘‘day-configuration’’ and a ‘‘night-configuration’’ The night-configuration is relatively insensitive to malate (the Ki for malate is 0.06—0.9 mM, depending on pH) and has a high affinity for PEP (the Km for PEP is 0.1—0.3 mM) The day-configuration is strongly inhibited by malate (the Kifor malate is 0.004—0.07 mM, again depending on the pH) and has a low affinity for PEP (the Kmfor PEP is 0.7—1.25 mM) Therefore, when

malate is rapidly exported to the vacuole at night in phase I, the carboxylation of PEP readily takes place, whereas it is suppressed during the day in phase III The modification of the kinetic properties involves the phosphorylation and de-phosphorylation of PEP carboxylase (Nimmo et al 2001)

Through modification of its kinetic properties, the inhibition of PEP carboxylase prevents a futile cycle of carboxylation and concomitant decarboxy-lation reactions Further evidence that such a futile cycle does not occur comes from studies on the labeling with13C of the first or fourth carbon atom in malate If a futile cycle were to occur, doubly labeled malate should appear, as fumarase in the mitochondria would randomize the label in the malate molecule Such randomization only occurs during the acidification phase, indicating rapid exchange of the malate pools of the cytosol and the mitochondria, before malate enters the vacuole

TABLE12 Cumulative daily net CO2and O2exchange in the dark and in the light periods (12 hours each) and the daily Photosynthetic Quotient for the entire 24 hours period of a shoot of Ananas comosus (pineapple).*

Cumulative daily net CO2and O2exchange (mmol shoot–1]

Dark Light

CO2assimilation O2consumption CO2assimilation O2release Daily Photosynthetic Quotient

Day 10.6 6.4 10.4 27.1 0.99

Day 11.1 6.3 10.7 27.5 0.98

Source: Cote´ et al (1989)

*Photosynthetic quotient is the ratio of the total net amount of O

2evolved to the net CO2fixed in 24 hours (i.e., the total

amount of O2evolved in the light period minus the total amount of O2consumed in the dark period) to the total amount of

CO2fixed in the light plus dark period Measurements were done over two consecutive days

FIGURE 46 CO2 fixation in

CAM plants, showing diurnal patterns for net CO2

Next to malate, glucose 6-phosphate is also an effector of PEP carboxylase (Table 13) The physio-logical significance of this effect is that glucose 6-phosphate, which is produced from glucose, dur-ing its conversion into PEP thus stimulates the car-boxylation of PEP

Temperature has exactly the opposite effect on the kinetic properties of PEP carboxylase from a CAM plant and that from a C4 plant (Fig 48) These temperature effects help to explain why a low temperature at night enhances acidification

10.3 Water-Use Efficiency

Since CAM plants keep their stomata closed during the day when the vapor pressure difference (wi—wa) between the leaves and the surrounding air is high-est, and open at night when wi—wa, is lowest, they have a very high water-use efficiency As long as they are not severely stressed which leads to

complete closure of their stomata, the WUE of CAM plants tends to be considerably higher than that of both C3and C4plants (Table in Chapter on plant water relations)

Populations of the leaf-succulent Sedum wrightii (Crassulaceae) differ greatly in their leaf thickness, 13C values (ranging from —13.8 to —22.9%), the pro-portion of day vs night CO2uptake, and growth The largest plants exhibit the greatest proportion of day vs night CO2 uptake and hence the lowest WUE, suggesting an inverse relation between the plants’ ability to conserve water and their ability to gain carbon (Kalisz & Teeri 1986)

10.4 Incomplete and Facultative CAM Plants

When exposed to severe desiccation, some CAM plants may not even open their stomata during the TABLE 13 Effects of malate and glucose-6-phosphate (G6P) on the kinetic

parameters of PEP carboxylase.*

Vmax Ratio Km Ratio

mmol mg–1(Chl) min–1 mM

Control 0.42 1.0 0.13 1.0

ỵ1 mM G6P 0.45 1.07 0.08 0.61

ỵ2 mM G6P 0.47 1.12 0.05 0.39

ỵ5 mM malate 0.31 0.74 0.21 1.60 ỵ5 mM malate and mM G6P 0.34 0.81 0.05 0.39

FIGURE47 Gas exchange of Ananas comosus

(pineap-ple) during the dark and light period O2consumption

during the day is measured using the stable isotope18O

Gross O2release is the sum of net O2production and 18O

2 consumption The phases are the same as those

shown in Fig 45 (after Cote´ et al 1989) Copyright American Society of Plant Biologists

night (Bastide et al 1993), but they may continue to show a diurnal fluctuation in malic acid concentra-tion, as first found in Opuntia basilaris (prickly pear) The CO2 they use to produce malic acid at night does not come from the air, but is derived from respiration It is released again during the day, allowing some Rubisco activity This metabolism is termed CAM idling Fluorescence measurements have indicated that the photosystems remain intact during severe drought CAM idling can be consid-ered as a modification of normal CAM The plants remain ‘‘ready to move’’ as soon as the environmen-tal conditions improve, but keep their stomata closed during severe drought

Some plants show a diurnal fluctuation in the concentration of malic acid without a net CO2 uptake at night, but with normal rates of CO2 assim-ilation during the day These plants are capable of recapturing most of the CO2 derived from dark respiration at night, and to use this as a substrate for PEP carboxylase This is termed CAM cycling (Patel & Ting 1987) In Peperomia camptotricha, 50% of

the CO2released in respiration during the night is fixed by PEP carboxylase At the beginning of the day, some of the CO2that is fixed at night becomes available for photosynthesis, even when the stoma-tal conductance is very low In Talinum calycinum (fame flower), naturally occurring on dry rocks, CAM cycling may reduce water loss by 44% CAM cycling enhances a plant’s water-use efficiency (Harris & Martin 1991)

CAM idling typically occurs in ordinary CAM plants that are exposed to severe water stress and have a very low stomatal conductance throughout the day and night CAM cycling occurs in plants that have a high stomatal conductance and normal C3 photosynthesis during the day, but refix the CO2 produced in dark respiration at night which ordin-ary C3plants lose to the atmosphere

In a limited number of species, CAM only occurs upon exposure to drought stress: facultative CAM plants For example, in plants of Agave deserti, Clusia uvitana, Mesembryanthemum crystallinum(ice plant), and Portulacaria afra (elephant’s foot), irrigation with saline water or drought can change from a virtually normal C3 photosynthesis to the CAM mode (Fig 49; Winter et al 1992) We know of one genus containing C4species that can shift from a normal C4 mode under irrigated conditions, to a CAM mode under water stress: Portulaca grandiflora (moss rose), Portulaca mundula (hairy purslane), and Portulaca oleracea (common purslane) (Koch & Kennedy 1982, Mazen 1996) The transition from the C3or C4 to the CAM mode coincides with an enhanced PEP carboxylase activity and of the mRNA encoding this enzyme Upon removal of NaCl from the root envir-onment of Mesembryanthemum crystallinum (ice plant), the level of mRNA encoding PEP carboxy-lase declines in to hours by 77% The amount of the PEP carboxylase enzyme itself declines more slowly: after to days the activity is half its original level (Vernon et al 1988)

10.5 Distribution and Habitat of CAM Species

CAM is undoubtedly an adaptation to drought, since CAM plants close their stomata during most of the day This is illustrated in a survey of epiphytic bromeliads in Trinidad (Fig 50) There are two major ecological groupings of CAM plants: succu-lents from arid and semi-arid regions and epi-phytes from tropical and subtropical regions (Ehleringer & Monson 1993) In addition, there are some submerged aquatic plants exhibiting CAM (Sect 11.5) Although CAM plants are uncommon

FIGURE48 The effect of temperature on kinetic

proper-ties of PEP carboxylase from leaves of a Crassula argenta (jade plant, a CAM plant) and Zea mays (corn, a C4

plant) (A) Effect on percent inhibition by mM malate (B) Effect on the inhibition constant (Ki) for malate (Wu

in cold environments, this may reflect their evolu-tionary origin in warm climates rather than a tem-perature sensitivity of the CAM pathway (Nobel & Hartsock 1990) Roots of some orchids which lack stomata also show CAM

In temperate regions and alpine habitats world-wide, CAM plants, or species showing incomplete or facultative CAM, occur on shallow soils and rock outcrops, niches that are rather dry in moist climates

10.6 Carbon-Isotope Composition of CAM Species

Like Rubisco from C3 and C4 plants, the enzyme from CAM plants discriminate against13CO2, but,

FIGURE49 Induction of CAM in

the facultative CAM species Mesembryanthemum crystalli-num (ice plant), growing in its natural habitat on rocky coastal cliffs of the Mediterranean Sea Upon prolonged exposure to drought, the leaf water content (A) declines, and the nocturnal malate concentration (B) increases (yellow symbols and bars, day; turquoise symbols and bars, night) There is a shift from the C3mode to CAM, coinciding

with less carbon-isotope fractio-nation (C) (Osmond et al 1982)

FIGURE50 The relationship between percentage of

epi-phytic bromeliad species with CAM in a tropical forest and mean annual rainfall across the north-south preci-pitation gradient in Trinidad (Winter & Smith 1996)

the fractionation at the leaf level is considerably less than that of C3 plants and similar to that of C4 species (Fig 44) This is expected, as the stomata are closed during malate decarboxylation and fixa-tion of CO2by Rubisco Hence, only a small amount of CO2diffuses back from the leaves to the atmo-sphere, and Rubisco processes the accumulated 13

CO2(Sects 9.3 and 9.4)

Upon a shift from C3to CAM photosynthesis in facultative CAM plants, the stomata are closed dur-ing most of the day and open at night, and the carbon-isotope fractionation decreases (Fig 49) Hence, the carbon-isotope composition of CAM plants can be used as an estimate of the employment of the CAM pathway during past growth

11 Specialized Mechanisms Associated with Photosynthetic Carbon Acquisition in Aquatic Plants

11.1 Introduction

Contrary to the situation in terrestrial plants, in submerged aquatic plants chloroplasts are fre-quently located in the epidermis In terrestrial plants, CO2 diffuses from the air through the sto-mata to the mesophyll cells In aquatic plants, where diffusion is directly through the outer epidermal cell walls, the rate of this process is often limiting for photosynthesis A thick boundary layer around the leaves, and slow diffusion of CO2in water limit the rate of CO2 uptake How aquatic plants cope with these problems? To achieve a reasonable rate of photosynthesis and avoid excessive photore-spiration, special mechanisms are required to allow sufficient diffusion of CO2 to match the requirement for photosynthesis Several specialized mechanisms have evolved in different species adapted to specific environmental conditions Another feature of the habitat of many submerged aquatics is the low irradiance Leaves of many aqua-tics have the traits typical of shade leaves (Sect 3.2)

11.2 The CO2Supply in Water

In fresh water, molecular CO2is readily available Between 10 and 208C, the partitioning coefficient (that is, the ratio between the molar concentration of CO2 in air and that in water) is about The equilibrium concentration in water at an atmo-spheric CO2 concentration of 380 mmol mol—1 is

12 mM (at 258C, but rapidly decreasing with increas-ing temperature) Under these conditions, leaves of submerged aquatic macrophytes experience about the same CO2 concentration as those in air The diffusion of dissolved gasses in water, however, occurs approximately 104 times more slowly than in air, leading to rapid depletion of CO2inside the leaf during CO2 assimilation In addition, the O2 concentration inside photosynthesizing leaves may increase Decreasing CO2concentrations, especially in combination with increasing O2, inexorably lead to conditions that restrict the carboxylating activity and favor the oxygenating activity of Rubisco (Mommer et al 2005)

The transport of CO2 through the unstirred boundary layeris only by diffusion The thickness of the boundary layer is proportional to the square root of the leaf dimension, measured in the direction of the streaming water, and inversely proportional to the flow of the streaming water (Sect 2.4 of Chap-ter 4A on the plant’s energy balance) It ranges from 10 mm in well stirred media, to 500 mm in nonstirred media The slow diffusion in the boundary layer is often a major factor limiting an aquatic macro-phyte’s rate of photosynthesis

CO2dissolved in water interacts as follows:

H2O ỵ CO2 , H2CO3

, Hỵỵ HCO3 , 2H ỵ

ỵ CO23 (10)

HCO3 , OH 

ỵ CO2 (11)

Since the concentration of H2CO3is very low in comparison with that of CO2, the two are commonly combined and indicated as [CO2]

The interconversion between CO2 and HCO3 is slow, at least in the absence of carbonic anhy-drase The presence of the dissolved inorganic car-bon compounds strongly depends on the pH of the water (Fig 51) In ocean water, as pH increases from 7.4 to 8.3, the contribution of dissolved inorganic carbon species shifts as follows: CO2as a fraction of the total inorganic carbon pool decreases from to 1%, that of HCO3from 96 to 89%, and that of CO32increases from 0.2 to 11%

CO2availability in water at a neutral pH While the concentration of all dissolved inorganic carbon (i.e., CO2, HCO3, and CO32) may decline by a few percent only, the CO2concentration declines much more, since the high pH shifts the equilibrium from CO2to HCO3(Fig 51) This adds to the diffusion problem and further aggravates the limitation by supply of inorganic carbon for assimilation in sub-merged leaves that only use CO2and not HCO3

11.3 The Use of Bicarbonate by Aquatic Macrophytes

Many aquatic macrophytes, cyanobacteria, and algae can use HCO3, in addition to CO2, as a carbon source for photosynthesis (Maberly & Mad-sen 2002) This might!be achieved either by active uptake of HCO3 itself, or by proton extrusion, commonly at the abaxial side of the leaf, thus low-ering the pH in the extracellular space and shifting the equilibrium towards CO2 (Elzenga & Prins 1988) In some species [e.g., Elodea canadensis (waterweed)] the conversion of HCO3 into CO2 is also catalyzed by an extracellular carbonic anhy-drase In Ranunculus penicillatus spp pseudofluitans (a stream water crowfoot), the enzyme is closely associated with the epidermal cell wall (Newman & Raven 1993) Active uptake of HCO3 also requires proton extrusion, to provide a driving force

Aquatic plants that use HCO3 in addition to CO2have a mechanism to concentrate CO2in their chloroplasts Although this CO2-concentrating mechanism differs from that of C4 plants (Sect 9.2), its effect is similar: it suppresses the oxygenat-ing activity of Rubisco and lowers the CO2 -compen-sation point In Elodea canadensis (common waterweed), Potamogeton lucens (ribbonweed), and other aquatic macrophytes, the capacity to acidify the lower side of the leaves, and thus to use HCO3, is expressed most at high irradiance and low dis-solved inorganic carbon concentration in the water (Elzenga & Prins 1989) The capacity of the carbon-concentrating mechanism also depends on the N supply: the higher the supply, the greater the capa-city of the photosynthetic apparatus as well as that of the carbon- concentrating mechanism (Madsen & Baattrup- Pedersen 1995) Acidification of the lower side of the leaves is accompanied by an increase in extracellular pH at the upper side of the leaves The leaves become ‘‘polar’’ when the carbon supply from the water is less than the CO2-assimilating capacity (Prins & Elzenga 1989) There are also anatomical differences between the upper and lower side of ‘‘polar’’ leaves: the lower epidermal cells are often transfer cells, characterized by ingrowths of cell-wall material which increases the surface area of the plasma membrane They contain numerous mitochondria and chloroplasts At the upper side of the leaves, the pH increase leads to precipitation of calcium carbonates This process plays a major role in the geological sedimentation of calcium car-bonate (Sect 11.7)

Due to the use of HCO3, the internal CO2 con-centration may become much higher than it is in terrestrial C3plants This implies that they not need a Rubisco enzyme with a high affinity for CO2 Interestingly, just like C4plants (Sect 9.4), they have a Rubisco with a relatively high Km for CO2 The values are approximately twice as high as those of terrestrial C3plants (Yeoh et al 1981) This high Km is associated with a high maximum catalytic activity (kcat) of Rubisco, as in the HCO3-using green alga, Chlamydomonas reinhardtii, and in C4species For the Rubisco of the cyanobacterium Synechococcus that also has a carbon-concentrating mechanism, even higher Km(CO2) and kcat values are reported (Table 9)

Hydrilla verticillata(waterthyme) has an induci-ble CO2-concentrating mechanism, even when the pH of the medium is so low that there is no HCO3 available This monocotyledonous species predates modern terrestrial C4monocots and may represent an ancient form of C4photosynthesis (Magnin et al 1997) The species has an inducible single-cell

FIGURE51 The contribution of the different inorganic

carbon species as dependent on the pH of the water (Osmond et al 1982)

C4-type photosynthetic cycle (Table 7; Sect 9.5) This mechanism is induced at high temperatures and when the plants are growing in water that contains low concentrations of dissolved inorganic carbon (Reiskind et al 1997) There appears to be a clear ecological benefit to this CO2-concentrating mechanism when the canopy becomes dense, the dissolved O2 concentration is high, and the CO2 supply is low Under these conditions photorespira-tion decreases photosynthesis of a C3-type plant by at least 35%, whereas in Hydrilla verticillata this decrease is only about 4% (Bowes & Salvucci 1989) A carbon-concentrating mechanism in the form of a single-cell C4-like pathway has also been identified in a marine diatom of common occurrence in the oceans (Reinfelder et al 2000), indicating that this pathway is more common than thought previously (Sage 2004)

11.4 The Use of CO2from the Sediment

Macrophytes like water lilies that have an internal ventilation system assimilate CO2arriving from the roots due to pressurized flow (Sect 4.1.4 of Chapter 2B on plant respiration) The use of CO2from the sediment is only minor for most emergent wetland species such as Scirpus lacustris (bull rush) and Cyperus papyrus (papyrus), where it approximates 0.25% of the total CO2 uptake in photosynthesis (Farmer 1996) For Stratiotes aloides (water soldier),

the sediment is a major source of CO2, although only after diffusion into the water column (Prins & de Guia 1986) Stylites andicola is a vascular land plant without stomata that derives nearly all its carbon through its roots (Keeley et al 1984)

Submerged macrophytes of the isoetid life form (quillworts) receive a very large portion of their carbon for photosynthesis directly from the sedi-ment via their roots: 60 to 100% (Table 14) This capability is considered an adaptation to growth in low-pH, carbon-poor (‘‘soft-water’’) lakes, where these plants are common None of the investigated species from ‘‘hard-water’’ lakes or marine systems show significant CO2uptake via their roots (Farmer 1996) In the quillworts, CO2diffuses from the sedi-ment, via the lacunal air system to the submerged leaves These leaves are thick with thick cuticles, have no functional stomata when growing sub-merged, but large air spaces inside, so that gas exchange with the atmosphere is hampered, but internal exchange is facilitated Emergent leaves have very few stomata at the leaf base, and normal densities at the leaf tips (Fig 52) The chloroplasts in isoetid leaves are concentrated around the lacunal system The air spaces in the leaves are connected with those in stems and roots, thus facilitating the transport of CO2from the sediment to the leaves where it is assimilated At night, only part of the CO2 coming up from the sediment via the roots through the lacunal system is fixed (Sect 11.5), the rest being lost to the atmosphere

TABLE 14 Assimilation of14CO2derived from the air or from the rhizosphere by leaves and roots of Littorella uniflora (quillwort).*

14

CO2assimilation[mg C g–1(leaf or root DM) h–1]

Leaves Roots

Source: Air Rhizosphere Air Rhizosphere

CO2concentration

around the roots (mM)

0.1 300 340 10 60

(10) (50) (0.3) (70)

0.5 350 1330 10 170

(5) (120) (0.3) (140)

2.5 370 8340 10 570

(4) (1430) (0.3) (300)

Source: Nielsen et al (1991)

* 14CO

2was added to the air around the leaves or to the water around the

11.5 Crassulacean Acid Metabolism (CAM) in Aquatic Plants

Though aquatic plants by no means face the same problems connected with water shortage as desert plants, some of them [Isoetes (quillwort) species] have a similar photosynthetic metabolism: Crassula-cean Acid Metabolism (CAM) (Keeley 1990) They accumulate malic acid during the night and have rates of CO2fixation during the night that are similar in magnitude as those during the day, when the CO2 supply from the water is very low (Fig 53) The aerial leaves of Isoetes howellii, in contrast to the submerged leaves of the same plants, not show a diurnal fluctuation in the concentration of malic acid

Why would an aquatic plant have a similar photosynthetic pathway as is common in species from arid habitats? CAM in Isoetes is considered an adaptation to very low levels of CO2in the water, especially during the day (Fig 53), and allows the plants to assimilate additional CO2 at night This nocturnal CO2fixation gives them access to a carbon source that is unavailable to other species Though some of the carbon fixed in malic acid comes from the surrounding water, where it accumulates due to the respiration of aquatic organisms, some is also derived from the plant’s own respiration during the night A CAM pathway has also been discovered in other genera of aquatic vascular plants (Maberly & Madsen 2002)

11.6 Carbon-Isotope Composition of Aquatic Plants

There is a wide variation in carbon-isotope compo-sition among different aquatic plants, as well as a

large difference between aquatic and terrestrial plants (Fig 54) A low carbon-isotope fractionation might reflect the employment of the C4pathway of photosynthesis, although the typical Kranz anat-omy is usually lacking Only about a dozen aquatic C4species have been identified, and very few have submersed leaves with a well developed Kranz anatomy (Bowes et al 2002) A low carbon-isotope fractionation in aquatic plants might also reflect the CAM pathway of photosynthesis Isoetids often have rather negative 13C values, due to the isotope composition of the substrate (Table 15) Four factors account for the observed variation in isotope com-position of freshwater aquatics (Keeley & Sandquist 1992):

1 The isotope composition of the carbon source varies substantially It ranges from a 13C value of þ1%, for HCO3derived from limestone, to —30%, for CO2 derived from respiration The average 13C value of CO2 in air is —8% The isotope composition also changes with the water depth (Table 16)

2 The species of inorganic carbon fixed by the plant; HCO3has a 13C that is 7—11% less nega-tive than that of CO2

3 Resistance for diffusion across the unstirred boundary layer is generally important (except in rapidly streaming water), thus decreasing car-bon-isotope fractionation (Box 2)

4 The photosynthetic pathway (C3, C4, and CAM) that represent different degrees of fractionation

The isotope composition of plant carbon is dominated by that of the source (see and above), because diffusional barriers are strong (see 3) This accounts for most of the variation as described in Fig 54, rather than biochemical differ-ences in the photosynthetic pathway (Osmond et al 1982)

11.7 The Role of Aquatic Macrophytes in Carbonate Sedimentation

The capacity of photosynthetic organisms [e.g., Chara (musk-grass), Potamogeton (pondweed), and Elodea(waterweed)] to acidify part of the apoplast and use HCO3(Sect 11.3) plays a major role in the formation of calcium precipitates in fresh water, on both an annual and a geological time scale Many calcium-rich lake sediments contain plant-induced carbonates, according to:

Ca2ỵỵ 2HCO3 ! CO2ỵ CaCO3 (12)

FIGURE52 The stomatal density along mature leaves of

Littorella uniflora (shoreweed) from the base to the tip (Nielsen et al 1991)

This reaction occurs in the alkaline compartment that is provided at the upper side of the polar leaves of aquatic macrophyytes (Sect 11.3) Similar amounts of carbon are assimilated in photosynth-esis and precipitated as carbonate If only part of the CO2 released in this process is assimilated by the

macrophyte, as may occur under nutrient-deficient conditions, CO2is released to the atmosphere On the other hand, if the alkalinity of the compartment is relatively low, there is a net transfer of atmo-spheric CO2 to the water (McConnaughey et al 1994)

FIGURE53 CAM photosynthesis in submerged leaves

of Isoetes howellii (quillwort) in a pool (A) Malic acid levels, (B) rates of CO2uptake, and (C) irradiance at

the water surface, water temperatures, and concen-trations of CO2and O2; the numbers near the symbols

Equation (12) shows how aquatic photosynthetic organisms play a major role in the global carbon cycle, even on a geological time scale On the other hand, rising atmospheric CO2concentrations have

an acidifying effect and dissolve part of the calcium carbonate precipitates in sediments, and thus con-tribute to a further rise in atmospheric [CO2] (Sect 12)

12 Effects of the Rising CO2

Concentration in the Atmosphere

Vast amounts of carbon are present in carbonates in the Earth’s crust Also stored in the Earth’s crust is another major carbon pool: the organic carbon derived form past photosynthesis; a key factor in the development of the present low CO2/high O2 atmosphere Some CO2enters the atmosphere when carbonates are used for making cement, but apart from that, carbonates are only biologically impor-tant on a geological time scale Far more imporimpor-tant for the carbon balance of the atmosphere is the burn-ing of fossil fuels (coal, oil, and natural gas) and changes in land-use that represent a CO2input into the atmosphere of 8.1015g of carbon per year (1015g equals petagram, Pg) Compared with the total amount of carbon present in the atmosphere, 720 Pg, such inputs are substantial and inevitably affect the CO2 concentration in the Earth’s atmo-sphere (Falkowski et al 2000) CO2is, by far, the largest contributor to the anthropogenically enhanced greenhouse effect (Houghton 2007)

Since the beginning of the industrial revolution in the late 18th century, the atmospheric CO2 con-centration has increased from about 290 mmol mol—1 to the current level of over 385 mmol mol—1 (Tans 2007) The concentration continues to rise by about TABLE 15 Carbon-isotope composition (d13C in ø)

of submerged and emergent Isoetes howellii plants.*

Pondwater carbonate –15.5 to –18.6 Submerged

Leaves –27.9 to –29.4 Roots –25.8 to –28.8 Emergent

Leaves –29.4 to –30.1 Roots –29.0 to –29.8

Source: Keeley & Busch (1984)

*

Values are given for both leaves and roots and also for the pondwater carbonate

TABLE 16 Changes in the dissolved carbon isotope composition with depth as reflected in the composi-tion of the organic matter at that depth

Water depth (m) d13

C (ø)

1 –20.80

2 –20.75

5 –23.40

7 –24.72

9 –26.79

11 –29.91

Source: Osmond et al (1982)

FIGURE54 Variation in the

car-bon-isotope composition (13C)

of freshwater and marine aqua-tic species The observed varia-tion is due to variavaria-tion in 13C

values of the substrate and in the extent of diffusional limitation (Osmond et al 1982)

FIGURE55 The rise in atmospheric CO2

concentration, as measured at Mauna Loa (Hawaii), accelerated from about 0.7 mmol mol–1yr–1in the early years to about 2.0 mmol mol–1yr–1today The blue line refers to data collected during 1958–1974 at the Scripps Institute of Oceanography; the red line refers to data collected since 1974 by the National Oceanic and Atmospheric Administration, US Department of Commerce (Tans 2007) Reproduced with the author’s permission

FIGURE56 The global carbon cycle and global carbon

reservoirs Units are Pg C or Pg C yr–1; petagram ¼ 1015g ¼ 109metric tones (updated following Houghton

1.5 mmol mol—1per year (Fig 55) Measurements of CO2concentrations in ice cores indicate a pre-indus-trial value of about 280 mmol mol—1during the past 10000 years, and about 205 mmol mol—1some 20000 years ago during the last ice age Considerable quantities of CO2have also been released into the atmosphere as a result of deforestation, ploughing of prairies, drainage of peats, and other land-use changes that cause oxidation of organic compounds in soil and, to a lesser extent, biomass Combustion of fossil fueladds far greater amounts of carbon per year (Fig 56) Combined anthropogenic fluxes to the atmosphere amount to Pg of carbon per year (Falkowski et al 2000) Yet, the increase in the atmosphere is only 4.2 Pg of carbon per year (2000—2005) About 2.2 Pg of the ‘‘missing’’ carbon is taken up in the oceans and a similar amount (2.3 Pg) is fixed by terrestrial ecosystems (Grace 2004, Houghton 2007) Analysis of atmospheric CO2 con-centrations and its isotopic composition shows that north-temperate and boreal forests are the most likely sinks for the missing carbon There is also strong uptake by tropical forests, but this is offset by CO2 release from deforestation in the tropics This increased terrestrial uptake of CO2has many causes, including stimulation of photosynthesis by elevated [CO2] (about half of the increased terres-trial uptake) or by N deposition in N-limited eco-systems and regrowth of northern and mid-latitude forests (Houghton 2007)

Since the rate of net CO2assimilation is not CO2 -saturated in C3 plants at 385 mmol mol—1CO2, the rise in CO2concentration is more likely to enhance photosynthesis in C3than in C4plants, where the rate of CO2assimilation is virtually saturated at a CO2concentration of 385 mmol mol—1(Bunce 2004) The consequences of an enhanced rate of photo-synthesis for plant growth are discussed in Sect 5.8 of Chapter on growth and allocation

12.1 Acclimation of Photosynthesis to Elevated CO2Concentrations

Upon long-term exposure to 700 mmol mol—1, about twice the present atmospheric CO2 concen-tration, there may be a reduction of the photosyn-thetic capacity, associated with reduced levels of Rubisco and organic N per unit leaf area This down-regulation of photosynthesis increases with increasing duration of the exposure to elevated [CO2] and is most pronounced in plants grown at low N supplies By contrast, water-stressed plants tend to increase net photosynthesis in

response to elevated [CO2] (Wullschleger et al 2002) Herbaceous plants consistently reduce sto-matal conductance in response to elevated [CO2], so that Cidoes not increase as much as would be expected from the increase in Ca, but their intrinsic WUEtends to be increased (Long et al 2004) Tree photosynthesis continues to be enhanced by ele-vated [CO2], except when seedlings are grown in small pots, inducing nutrient limitation (Norby et al 1999) The decrease in stomatal conductance of C3 plants often indirectly stimulates photosynth-esis in dry environments by reducing the rate of soil drying and therefore the water limitation of photosynthesis (Hungate et al 2002) C3 and C4 plants, however, benefit equally from increased water-use efficiency and water availability, redu-cing the relative advantage that C3 plants gain from their greater CO2 responsiveness of photo-synthesis (Wand et al 1999, Sage & Kubien 2003) Why would acclimation of photosynthesis to elevated [CO2] be more pronounced when N sup-ply is poor? This could be a direct effect of N or an indirect effect by limiting the development of sinks for photoassimilates This question can be tested by growing Lolium perenne (perennial ryegrass) in the field under elevated and current atmospheric CO2concentrations at both low and high N supply Cutting of this herbage crop at regular intervals removes a major part of the canopy, decreasing the ratio of photosynthetic area to sinks for photoas-similates Just before the cut, when the canopy is relatively large, growth at elevated [CO2] and low N supply decreases in carboxylation capacity and the amount of Rubisco protein At a high N supply there are no significant decreases in carboxylation capacity or proteins Elevated [CO2] results in a marked increase in leaf carbohydrate concentra-tion at low N supply, but not at high N supply This acclimation at low N supply is absent after a harvest, when the canopy size is small Acclima-tion under low N is therefore most likely caused by limitation of sink development rather than being a direct effect of N supply on photosynthesis (Rogers et al 1998)

How herbaceous plants sense that they are growing at an elevated CO2 concentration and then down-regulate their photosynthetic capacity? Acclimation is not due to sensing the CO2 concen-tration itself, but sensing the concenconcen-tration of sugars in the leaf cells, more precisely the soluble hexose sugars (Sect 4.2), mediated by a specific hexokinase (Sect 4.3) In transgenic plants in which the level of hexokinase is greatly reduced, down-regulation of photosynthesis upon pro-longed exposure to high [CO2] is considerably

less Via a signal-transduction pathway, which also involves phytohormones, the sugar-sensing mechanism regulates the transcription of nuclear encoded photosynthesis-associated genes (Rolland et al 2006) Among the first photosynthetic pro-teins that are affected are the small subunit of Rubisco and Rubisco activase Upon longer expo-sure, the level of thylakoid proteins and chloro-phyll is also reduced (Table 17)

The down-regulation of photosynthesis at ele-vated CO2has led to the discovery of sugar-sensing in plants, but it has recently become clear that the signaling pathway is intricately involved in a net-work regulating acclimation to other environmental factors, including light and nutrient availability as well as biotic and abiotic stress (Rolland et al 2002) Down-regulation of photosynthesis in response to long-term exposure to elevated CO2has important global implications The capacity of terrestrial eco-systems to sequester carbon appears to be saturat-ing, leaving a larger proportion of human carbon emissions in the atmosphere, and accelerating the rate of global warming (Canadell et al 2007)

12.2 Effects of Elevated CO2

on Transpiration—Differential Effects on C3, C4, and CAM Plants

Different types of plants respond to varying degrees to elevated CO2 For example, C4plants, whose rate of photosynthesis is virtually saturated at 385 mmol mol—1, generally respond less to elevated CO2than C3plants

Also Opuntia ficus-indica (prickly pear), a CAM species cultivated worldwide for its fruits and cla-dodes, responds to the increase in CO2 concentra-tion in the atmosphere The rate of CO2assimilation is initially enhanced, both at night and during the day, but this disappears upon prolonged exposure to elevated CO2(Cui & Nobel 1994) CAM species show, on average, a 35% increase in net daily CO2 uptake which reflects increases in both Rubisco-mediated CO2uptake during the day and PEP car-boxylase-mediated CO2uptake at night (Drennan & Nobel 2000)

13 Summary: What Can We Gain from Basic Principles and Rates of Single-Leaf Photosynthesis?

Numerous examples have been given on how dif-ferences in photosynthetic traits enhance a geno-type’s survival in a specific environment These include specific biochemical pathways (C3, C4, and CAM) as well as more intricate differences between sun and shade plants, aquatic and terrestrial plants, and plants differing in their photosynthetic N-use efficiency and water-use efficiency Information on photosynthetic traits is also highly relevant when trying to understand effects of global environmental changes in temperature and atmospheric CO2 con-centrations For a physiological ecologist, a full appreciation of the process of leaf photosynthesis is quintessential

What we cannot derive from measurements on photosynthesis of single leaves is what the rate of photosynthesis of an entire canopy will be To TABLE17 Light-saturated rate of photosynthesis (Amax, measured at the CO2concentrations at which the plants were grown), in vitro Rubisco activity, chlorophyll concentration and the concentration of hexose sugars in the fifth leaf of Solanum lycopersicum (tomato) at various stages of development.*

Amax(mmol m–2

s–1)

Rubisco activity (mmol m–2 s–1)mg m–2

Chlorophyll (mg m–2)

Glucose

(mg m–2) Fructose Leaf

expansion (% of full

Exposure time

expansion) (days) Control High Control High Control High Control High Control High

2 16.3 21.3 22.6 – 270 – 750 – 500 –

60 11 18.9 28.7 20.5 25.1 480 520 1000 1200 1400 1400 95 22 15.0 25.1 15.7 12.7 540 500 1100 1250 1800 2100 100 31 9.3 18.0 9.5 4.9 450 310 1100 2100 1800 4200

Source: Van Oosten & Besford (1995), Van Oosten et al (1995)

*

work out these rates, we need to take the approach discussed in Chapter 5, dealing with scaling-up principles It is also quite clear that short-term measurements on the effect of atmospheric CO2concentrations are not going to tell us what will happen in the long term Acclimation of the photosynthetic apparatus (‘‘down-regulation’’) may occur, reducing the initial stimulatory effect Most importantly, we cannot derive plant growth rates or crop yields from rates of photosynthesis of a single leaf Growth rates are not simply deter-mined by rates of single-leaf photosynthesis per unit leaf area, but also by the total leaf area per plant and by the fraction of daily produced photo-synthates required for plant respiration, issues that are dealt with in later chapters

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Chapter 2

Photosynthesis, Respiration, and Long-Distance Transport

2B Respiration

1 Introduction

A large portion of the carbohydrates that a plant assimilates each day are expended in respiration in the same period (Table 1) If we seek to explain the carbon balance of a plant and to understand plant performance and growth in different environments, it is imperative to obtain a good understanding of respiration Dark respiration is needed to produce the energy and carbon skeletons to sustain plant growth; however, a significant part of respiration may proceed via a nonphosphorylating pathway that is cyanide resistant and generates less ATP than the cytochrome pathway, which is the primary energy-producing pathway in both plants and ani-mals We present several hypotheses in this chapter to explore why plants have a respiratory pathway that is not linked to ATP production

The types and rates of plant respiration are controlled by a combination of respiratory capacity, energy demand, substrate availability, and oxygen supply (Covey-Crump et al 2002, 2007) At low levels of O2, respiration cannot proceed by normal aerobic pathways, and fermentation starts to take place, with ethanol and lactate as major end-products The ATP yield of fermentation is considerably less than that of normal aerobic respiration In this chapter, we discuss the control over respiratory processes, the demand for respiratory energy, and the significance of

respiration for the plant’s carbon balance, as these are influenced by species and environment

2 General Characteristics of the Respiratory System

2.1 The Respiratory Quotient

The respiratory pathways in plant tissues include glycolysis, which is located both in the cytosol and in the plastids, the oxidative pentose phosphate pathway, which is also located both in the plastids and the cytosol, the tricarboxylic acid (TCA) or Krebs cycle, in the matrix of mitochondria, and the electron-transport pathways, which reside in the inner mitochondrial membrane

respiration to fatty acids (Tcherkez et al 2003) For roots of young seedlings, measured in the absence of an N source, values close to 1.0 have been found, but most experimental RQ values differ from unity (Table 2) RQ values for germinating seeds depend on the sto-rage compounds in the seeds For seeds of Triticum aestivum (wheat), in which carbohydrates are major storage compounds, RQ is close to unity, whereas for the fat-storing seeds of Linum usitatissimum (flax) RQ values as low as 0.4 are found (Stiles & Leach 1936)

Both the nature of the respiratory substrate and biosynthetic reactions strongly influence RQ The RQ can be greater than 1, if organic acids are an important substrate, because these are more oxi-dized than sucrose, and, therefore, produce more CO2 per unit O2 On the other hand, RQ will be less than 1, if compounds that are more reduced than sucrose (e.g., lipids and protein) are a major substrate, as occurs during starvation of leaves and excised root tips (Table 2) In shoots of Hordeum vulgare (barley) that receive NH4+ as their sole N source respiratory fluxes of O2equal those of CO2 By contrast, shoots exposed to NO3-show a higher CO2evolution than O2consumption in the dark (RQ ¼ 1.25) These results show that a substantial por-tion of respiratory electron transport generates reductant for NO3 assimilation, producing an additional two molecules of CO2per molecule of NO3reduced to NH4ỵ (Bloom et al 1989) Sub-strates available to support root respiration depend on processes occurring throughout the plant For

example, organic acids (malate) that are produced during the reduction of NO3in leaves can be trans-ported and decarboxylated in the roots, releasing CO2 and increasing RQ (Ben Zioni et al 1971) If NO3-reductionproceeds in the roots, then the RQ is also expected to be greater than Values of RQ are therefore lower in plants that use NH4ỵas an N source than in plants grown with NO3or, symbio-tically, with N2(Table 2)

Biosynthesis influences RQ in several ways Car-boxylating reactions consume CO2, reducing RQ, whereas decarboxylating reactions produce CO2and, therefore, increase RQ In addition, synthesis of oxi-dized compounds such as organic acids decreases RQ, whereas the production of reduced compounds such as lipids leads to higher RQ values The average mole-cular formula of the biochemical compounds typical of plant biomass is more reduced than sucrose, so RQ values influenced by biosynthesis should be greater than 1, as generally observed (Table 2; for further information, see Table 5.11 in Sect 5.2.2)

RQ values of root respiration increase with increas-ing potential growth rate of a species (Fig 1) This results from high rates of biosynthesis, relative to rates of ATP production; as explained above, ATP production associated with sucrose breakdown is TABLE Utilization of photosynthates in plants, as

dependent on the nutrient supply.*

Item

Utilization of photosynthates % of C fixed

Free nutrient availability

Limiting nutrient supply

Shoot growth 40*–57 15–27* Root growth 17–18* 33*–35 Shoot

respiration

17–24* 19–20*

Root respiration 8–19* 38*–52 – Growth 3.5–4.6* 6*–9 – Maintenance 0.6–2.6* ? – Ion acquisition –13* ? Volatile losses 0–8 0–8 Exudation <5 <23 N2fixation Negligible 5–24

Mycorrhiza Negligible 7–20

Source : Van der Werf et al (1994)

* inherently slow-growing species; ? no information for nutrient-limited conditions

TABLE The respiratory quotient (RQ) of root respiration of a number of herbaceous species.*

Species RQ

Special Remarks

Allium cepa 1.0 Root tips 1.3 Basal parts Dactylis glomerata 1.2

Festuca ovina 1.0 Galinsoga parviflora 1.6 Helianthus annuus 1.5 Holcus lanatus 1.3 Hordeum distichum 1.0 Lupinus albus 1.4

1.6 N2-fixing

Oryza sativa 1.0 NH4ỵ-fed

1.1

Pisum sativum 0.8 NH4ỵ-fed

1.0

1.4 N2-fixing

Zea mays 1.0 Fresh tips 0.8 Starved tips

Source : Various authors, as summarized in Lambers et al (2002)

*All plants were grown in nutrient solution, with nitrate as

the N-source, unless stated otherwise The Pisum sativum (pea) plants were grown with a limiting supply of combined N, so that their growth matched that of the symbiotically grown plants

associated with an RQ of 1.0, whereas biosynthesis yields RQ values greater than 1.0 (Scheurwater et al 2002)

In summary, the patterns of RQ in plants clearly demonstrate that in roots it depends on the plant’s growth rate For all organs, it depends on the predo-minant respiratory substrate, integrated whole-plant processes, and ecological differences among species

2.2 Glycolysis, the Pentose Phosphate Pathway, and the Tricarboxylic (TCA) Cycle

The first step in the production of energy for respira-tion occurs when glucose (or starch or other storage carbohydrates) is metabolized in glycolysis or in the oxidative pentose phosphate pathway (Fig 2) Gly-colysisinvolves the conversion of glucose, via phos-phoenolpyruvate (PEP), into malate and pyruvate In contrast to mammalian cells, where virtually all PEP is converted into pyruvate, in plant cells malate is the major end-product of glycolysis and thus the major substrate for the mitochondria Key enzymes in glycolysis are controlled by adenylates (AMP, ADP, and ATP), in such a way as to speed up the rate of glycolysis when the demand for metabolic energy (ATP) increases (Plaxton & Podesta´ 2006)

Oxidation of one glucose molecule in glycolysis produces two malate molecules, without a net pro-duction of ATP When pyruvate is the end product, there is a net production of two ATP molecules in glycolysis Despite the production of NADH in one step in glycolysis, there is no net production of NADH when malate is the end product, due to the

need for NADH in the reduction of oxaloacetate, catalyzed by malate dehydrogenase

Unlike glycolysis, which is predominantly involved in the breakdown of sugars and ultimately in the production of ATP, the oxidative pentose phosphate pathwayplays a more important role in producing intermediates (e.g., amino acids, nucleo-tides) and NADPH There is no evidence for a con-trol of this pathway by the demand for energy

The malate and pyruvate that are formed in gly-colysis in the cytosol are imported into the mito-chondria, where they are oxidized in the tricarboxylic acid (TCA) cycle Complete oxidation of one molecule of malate, yields three molecules of CO2, five molecules of NADH and one molecule of FADH2, as well as one molecule of ATP (Fig 2) NADH and FADH2subsequently donate their elec-trons to the electron-transport chain (Sect 2.3.1)

2.3 Mitochondrial Metabolism

The malate formed in glycolysis in the cytosol is imported into the mitochondria and oxidized partly via malic enzyme, which produces pyruvate and CO2, and partly via malate dehydrogenase, which produces oxaloacetate Pyruvate is then oxidized so that malate is regenerated (Fig 2) In addition, pyr-uvate can be produced in the cytosol and imported into the mitochondria Oxidation of malate, pyru-vate, and other NAD-linked substrates is associated with complex I (Sect 2.3.1) In mitochondria there are four major complexes associated with electron transfer and one associated with oxidative phos-phorylation, all located in the inner mitochondrial membrane In addition, there are two small redox molecules, ubiquinone (Q) and cytochrome c, which play a role in electron transfer In plant mito-chondria there is also a cyanide-resistant, nonpho-sphorylating, alternative oxidase, located in the inner membrane (Fig 3) Finally, there are addi-tional NAD(P)H dehydrogenases in the inner mito-chondrial membrane that allow electron transport without ATP formation as well as uncoupling pro-teinsthat converts energy that could have been used for ATP production into heat

In the mitochondrial matrix the imported sub-strates are oxidized in a cyclical process (Krebs or TCA cycle), releasing three CO2molecules per pyr-uvate in each cycle and generating reducing power (NADH and FADH2) in several reactions (Fig 2) Pyruvate decarboxylase (PDC), which converts pyr-uvate into acetylCoA, which then reacts with oxa-loacetate to produce citrate, is a major control point for entry of carbon into the TCA cycle

FIGURE1 The respiratory quotient of a number of

2.3.1 The Complexes of the Electron-Transport Chain

Complex Iis the main entry point of electrons from NADH produced in the TCA cycle or in photore-spiration (glycine oxidation) Complex I is the first coupling siteor site of proton extrusion from the matrix into the intermembrane space which is linked to ATP production Succinate is the only intermediate of the TCA cycle that is oxidized by a membrane-bound enzyme: succinate dehydrogenase (Fig 3) Electrons enter the respiratory chain via complex II and are transferred to ubiquinone NAD(P)H that is produced outside the mitochondria also feeds its electrons into the chain at the level of ubiquin-one (Fig 3) As with complex II, the external

dehydrogenases are not connected with the trans-location of Hỵ across the inner mitochondrial membrane Hence less ATP is produced per O2 when succinate or NAD(P)H are oxidized in com-parison with that of glycine, malate, or citrate, which enter at complex I Complex III transfers electrons from ubiquinone to cytochrome c, coupled to the extrusion of protons to the inter-membrane space and is therefore site of proton extrusion from the matrix into the intermembrane space Complex IV is the terminal oxidase of the cytochrome pathway, accepting electrons from cytochrome c and donating these to O2 It also gen-erates a proton-motive force (i.e., an electrochemi-cal potential gradient across a membrane), which makes complex IV site of proton extrusion

FIGURE2 The major substrates for the electron transport

pathways Glycine is only a major substrate in

photosynthetically active cells of C3plants when

photo-respiration plays a role

2.3.2 A Cyanide-Resistant Terminal Oxidase

Mitochondrial respiration of many tissues from higher plants is not fully inhibited by inhibitors of the cytochrome path (e.g., KCN) This is due to the presence of a cyanide-resistant, alternative electron-transport pathway, consisting of one enzyme, the alternative oxidase, firmly embedded in the inner mitochondrial membrane The branching point of the alternative path from the cytochrome path is at the level of ubiquinone, a component common to both pathways Transfer of electrons from ubiqui-none to O2via the alternative path is not coupled to the extrusion of protons from the matrix to the inter-membrane space Hence, the transfer of electrons from NADH produced inside the mitochondria to O2 via the alternative path bypasses two sites of proton extrusion, and therefore yields only one

third of the amount of ATP that is produced when the cytochrome path is used

2.3.3 Substrates, Inhibitors, and Uncouplers

Figure summarizes the major substrates for mito-chondrial O2uptake as well as their origin Oxida-tion of glycine is of quantitative importance only in tissues exhibiting photorespiration Glycolysis may start with glucose, as depicted here, or with starch, sucrose, or any major transport carbohydrate or sugar alcohol imported via the phloem (Sect of Chapter 2C on long-distance transport)

A range of respiratory inhibitors have helped to elucidate the organization of the respiratory path-ways To give just one example, cyanide effectively blocks complex IV and has been used to demon-strate the presence of the alternative path

FIGURE3 The organization of the electron-transporting

complexes of the respiratory chain in higher plant chondria All components are located in the inner mito-chondrial membrane Some of the components are membrane spanning, others face the mitochondrial matrix or the space between the inner and the outer mitochondrial membrane Q (ubiquinone) is a mobile

Uncouplersmake membranes, including the inner mitochondrial membrane, permeable to protons and hence prevent oxidative phosphorylation Many compounds that inhibit components of the respiratory chain or have an uncoupling activity occur naturally as secondary compounds in plant and fungal tissues; they may protect these tissues from being grazed or infected by other organisms or be released from roots and act as allelochemicals (Sects and 3.1 of Chapter 9B on ecological bio-chemistry) A more recent addition to the complex-ity of the plant mitochondrial electron-transport chain is the discovery of uncoupling protein (UCP) (Hourton-Cabassa et al 2004) UCP is a homologue of thermogenin, a protein responsible for thermogenesis in mammalian brown fat cells Both uncoupling protein and thermogenin allow protons to diffuse down their concentration gradi-ent from the intermembrane space into the matrix, circumventing the ATP synthase complex and thus uncoupling electron transport from ATP production (Plaxton & Podesta´ 2006)

2.3.4 Respiratory Control

To learn more about the manner in which plant respiration responds to the demand for metabolic energy, we first describe some experiments with iso-lated mitochondria Freshly isoiso-lated intact mitochon-dria in an appropriate buffer that lacks substrates, a condition referred to as ‘‘state 1’’, not consume an appreciable amount of O2; in vivo they rely on a con-tinuous import of respiratory substrate from the cyto-sol (Fig 4) Upon addition of a respiratory substrate (‘‘state 2’’) there is some, but still not much O2uptake; for rapid rates of respiration to occur in vivo, import of additional metabolites is required As soon as ADP is added, a rapid consumption of O2can be measured This ‘‘state’’ of the mitochondria is called ‘‘state 3’’ In vivo, rapid supply of ADP will occur when a large amount of ATP is required to drive biosynthetic and transport processes Upon conversion of all ADP into ATP (‘‘state 4’’), the respiration rate of the mitochondria declines again to the rate found before addition of ADP (Fig 4) Upon addition of

FIGURE The ‘‘states’’ of isolated mitochondria The

ADP:O ratio (also called ATP:O ratio or P:O ratio) is calculated from the O2consumption during the

phos-phorylation of a known amount of added ADP (state 3) The amount of ADP consumed equals the amount that has been added to the cuvette (310 and 232 in A and B, respectively); since the total amount of O2in the cuvette

is known (300 nmol), the amount consumed during the consumption of the added ADP can be derived (dashed

vertical lines, with values of 183 and 89 nanomoles of O atoms in A and B, respectively) The respiratory control ratio (RC) is the ratio of the rate of O2uptake (in nmol O2

mg1protein min1; values written along the slopes) in

state and state State refers to the respiration in the absence of respiratory substrate and ADP, and state is the respiration after addition of respiratory substrate, but before addition of ADP (based on unpublished data from A.M Wagner, Free University of Amsterdam)

more ADP, the mitochondria go into state again, followed by state upon depletion of ADP This can be repeated until all O2in the cuvette is consumed Thus the respiratory activity of isolated mitochondria is effectively controlled by the availability of ADP: respiratory control, quantified in the ‘‘respiratory control ratio’’ (the ratio of the rate at substrate satura-tion in the presence of ADP and that under the same conditions, but after ADP has been depleted; Fig 4) The same respiratory control occurs in intact tissues and is one of the mechanisms ensuring that the rate of respiration is enhanced when the demand for ATP increases

2.4 A Summary of the Major Points of Control of Plant Respiration

We briefly discussed the control of glycolysis by ‘‘energy demand’’ (Sect 2.2) and a similar control by ‘‘energy demand’’ of mitochondrial electron transport, termed respiratory control (Sect 2.3.4) The effects of energy demand on dark respiration are a function of the metabolic energy that is required for growth, maintenance, and transport processes; therefore, when tissues grow quickly, take up ions rapidly and/or have a fast turnover of proteins, they generally have a high rate of respira-tion At low levels of respiratory substrate supply (carbohydrates, organic acids), however, the activity of respiratory pathways may be substrate-limited When substrate levels increase, the respiratory capacity is enhanced and adjusted to the high sub-strate input, through the transcription of specific genes that encode respiratory enzymes Figure summarizes these and several other points of con-trol Plant respiration is clearly quite flexible and responds rapidly to the demand for respiratory energy as well as the supply of respiratory substrate The production of ATP which is coupled to the oxidation of substrate, may also vary widely, due to the presence of both nonphosphorylating and phosphorylating paths [alternative oxidase and NAD(P) dehydrogenases other than complex I] as well as the activity of an uncoupling protein

2.5 ATP Production in Isolated Mitochondria and In Vivo

The rate of O2consumption during the phosphor-ylation of ADP can be related to the total ADP that must be added to consume this O2 This allows calculation of the ADP:O ratio in vitro This ratio is around 2.5 for NAD-linked substrates (e.g.,

malate, citrate) and around 1.5 for succinate and external NAD(P)H Nuclear Magnetic Resonance (NMR) spectroscopy has been used to estimate ATP production in intact tissues, as outlined in Sect 2.5.2

2.5.1 Oxidative Phosphorylation: The Chemiosmotic Model

During the transfer of electrons from various sub-strates to O2via the cytochrome path, protons are extruded into the space between the inner and outer mitochondrial membranes This generates a proton-motive forceacross the inner mitochondrial mem-brane which drives the synthesis of ATP The basic features of this chemiosmotic model are (Mitchell 1966, Nicholls & Ferguson 1992):

1 Protons are transported outwards, coupled to the transfer of electrons, thus giving rise to both a proton gradient(pH) and a membrane poten-tial()

2 The inner membrane is impermeable to protons and other ions, except by special transport systems There is an ATP synthetase (also called ATPase), which transforms the energy of the electrochemical gradient generated by the proton-extruding system into ATP

The pH gradient, pH, and the membrane potential , are interconvertible It is the combina-tion of the two which forms the proton-motive force (p), the driving force for ATP synthesis, catalyzed by an ATPase:

p ¼  2:3 RT=FpH (1)

where R is the gas constant (J mol1K1), T is the absolute temperature (K) and F is Faraday’s number (Coulomb) Both components in the equation are expressed in mV Approximately one ATP is pro-duced per three protons transported

2.5.2 ATP Production In Vivo

field The location of the peaks in a NMR spectrum depends on the molecule in which the nucleus is present and also on the ‘‘environment’’ of the mole-cule (e.g., pH) Figure illustrates this point for a range of phosphate — containing molecules (Roberts 1984)

The resonance of specific P — containing com-pounds can be altered by irradiation with radiofre-quency power If this irradiation is sufficiently strong (‘‘saturating’’), then it disorientates the nuclear spins of that P — containing compound, so that its peak disappears from the spectrum

FIGURE A simplified scheme of respiration and its

major control points Controlling factors include the concentration of respiratory substrate [e.g., glucose (1)] and adenylates (2, 3) Adenylates may exert control on electron transport via a constraint on the rate of oxidative phosphorylation (2) as well as on glycolysis, via modulation of the activity of key enzymes in glyco-lysis, phosphofructokinase and pyruvate kinase (‘‘energy demand’’, 3) When the input of electrons into the respiratory chain is very high, a large fraction of ubiqui-none becomes reduced and the alternative path becomes more active (4) When the rate of glycolysis is

very high, relative to the activity of the cytochrome path, organic acids may accumulate (5, 6) The accumu-lation of citric acid may lead to the reduction of the sulfide bonds of the alternative oxidase and thus enhance the capacity of the alternative path (5) Accu-mulation of pyruvate or other a-keto acids may increase the Vmaxof the alternative oxidase and, hence, allow it

to function at a low level of reduced ubiquinone (6) There is increasing evidence that the nonphosphorylat-ing rotenone-insensitive bypass (7) operates in concert with the alternative path, when the concentration of NADH is very high

Figure 7A illustrates this for the g-ATP P-atom, the P atom that is absent in ADP Upon hydrolysis of ATP, the g-ATP P atom becomes part of the cytoplasmic inorganic phosphate (Pi) pool For a brief period, therefore, some of the Pi molecules also contain disoriented nuclear spins; specific radiation of the g-ATP peak decreases the Pipeak This phenomenon is called ‘‘saturation transfer’’ (Fig 7) Saturation transfer has been used to estimate the rate of ATP hydrolysis to ADP and Piin vivo

If the rate of disappearance of the saturation in the absence of biochemical exchange of phosphate between g-ATP and Piis known, then the rate of ATP hydrolysis can be derived from the rate of loss of saturation This has been done for root tips for which the O2 uptake was measured in parallel experiments In this manner ADP:O ratios in Zea mays(maize) root tips exposed to a range of condi-tions have been determined (Table 3)

The ADP:O ratios for the root tips supplied with 50 mM glucose are remarkably close to those expected when glycolysis plus TCA cycle are responsible for the complete oxidation of exogenous glucose, provided the alternative path does not contribute to the O2 uptake (Table 3) KCN decreases the ADP:O ratio of glucose oxidation by two-thirds in a manner to be expected from mitochondrial studies SHAM, an inhi-bitor of the alternative path, has no effect on the rate of ATP production So far, maize root tips are the only

intact plant material used for the determination of ADP:O ratios in vivo We cannot assume, therefore, that the ADP:O ratio in vivo is invariably In fact, the ratio under most circumstances is probably far less than (Sect 2.6.2)

2.6 Regulation of Electron Transport via the Cytochrome and the Alternative Paths

The existence of two respiratory pathways, both transporting electrons to O2, in higher plant mito-chondria, raises the question if and how the parti-tioning of electrons between the two paths is regulated This is important because the cytochrome path is coupled to proton extrusion and the produc-tion of ATP, whereas transport of electrons via the alternative path is not, at least not beyond the point where both pathways branch to O2 (Millenaar & Lambers 2003)

2.6.1 Competition or Overflow?

Under specific conditions, the activity of the cyto-chrome path in vitro increases linearly with the

FIGURE6 NMR spectrum of root tips of Pisum sativum

(pea), showing peaks of, for example, glucose-6-phos-phate (1), (Pi) (2, 3), and ATP (4, 5) in a living plant cell

The exact radiofrequency at which a phosphate-contain-ing compound absorb depends on the pH This explains why there are two peaks for Pi: a small one for the

cytosol (2), where the pH is approximately 7, and a larger one for the vacuole (3), where the pH is lower (Roberts 1984) Reprinted, with permission, from the Annual Review of Plant Physiology, Volume 35 #1984 by Annual Reviews www.annualreviews.org

FIGURE7 Saturation transfer from g -ATP phosphate to

cytosolic Piin root tips of Zea mays (maize) Spectrum A

was obtained with selective presaturation of the g -ATP peak Spectrum B was obtained with selective presatura-tion of a point equidistant from the cytosolic Pipeak,

Spectrum A–B gives the difference between the two spectra, showing the transfer of saturation from g -ATP to cytosolic Pi(after Roberts et al 1984a) Copyright

fraction of ubiquinone (Q, the common substrate with the alternative path) that is in its reduced state (Qr/Qt) By contrast, the alternative path shows no appreciable activity until a substantial (30—40%) fraction of the Q is in its reduced state, and then the activity increases exponentially (Fig 8) This would suggest that the alternative path functions as an ‘‘energy overflow’’; however,

recent experimental results suggest that this is an over-simplification, as outlined below

2.6.2 The Intricate Regulation of the Alternative Oxidase

Depending on metabolic state, the activity of the alternative pathway changes, so that it competes with the cytochrome pathway for electrons When embedded in the inner mitochondrial membrane, the alternative oxidase exists as a dimer, with the two subunits linked by disulfide bridges These sulfide bridges may be oxidized or reduced If they are reduced, then the alternative oxidase is in its higher-activity state, as opposed to the lower-activ-ity state when the disulfide bridges are oxidized Roots of soybean seedlings (Glycine max) initially have a very high respiration rate, and almost all of this respiration occurs via the cytochrome path (Fig 9A) At this stage, the activity of the alternative path is very low and the enzyme is in its oxidized (lower-activity) state Within a few days, the growth rate and the cytochrome oxidase activity decline about fourfold, and the contribution of the alterna-tive path to root respiration increases to more than 50% At that stage, all the dimers are in their reduced (higher-activity) state, suggesting that the transition from partly oxidized to fully reduced is responsible for the increased alternative oxidase activity (Millar et al 1998) A similar change from oxidized to reduced occurs in leaves of Alocasia odora (Japanese taro) upon exposure to high-light stress, as dis-cussed in Section 4.4 In intact roots of Poa annua (annual meadow-grass) and several other grasses, however, the alternative oxidase is invariably in its reduced, higher-activity configuration (Millenaar et al 1998, 2000) There is, therefore, no clear evi-dence that changes in redox state of the alternative TABLE3 The in vivo ADP:O ratios in root tips of Zea mays (corn) determined with the saturation transfer31P NMR technique and O2uptake measurements

Exogenous substrate O2concentration Inhibitor Rate of O2uptake Rate of ATP production ADP:O ratio

Glucose 100 None 22 143 3.2

Glucose None <20 –

None 100 None 15 93 3.0

Glucose 100 KCN 14 26 1.0

Glucose 100 KCN+SHAM <20 –

Glucose 100 SHAM 21 137 3.2

Source: Roberts et al (1984a)

*The O

2concentration was either that in air (100) or zero Rates of ATP production and O2consumption are expressed as

nmol g1FM s1 Exogenous glucose was supplied at 50 mM The concentration of KCN was 0.5 mM and that of SHAM was

2 mM; this is sufficiently high to fully block the alternative path in maize root tips

FIGURE8 Dependence of the activity of the cytochrome

path and of the alternative path on the fraction of ubiqui-none that is in its reduced state (Qr/Qt) When the

alter-native oxidase is in its ‘‘reduced’’ (higher-activity) configuration, it has a greater capacity to accept elec-trons In its reduced state, the alternative oxidase can be affected by a-keto acids, which enhance its activity at low levels of Qr [Based on Dry et al (1989), Umbach et al

(1994), Day et al (1995), and Hoefnagel et al (1997)]

oxidase play an important regulatory role in vivo during plant development (Hoefnagel & Wiskich 1998)

The alternative oxidase’s capacity to oxidize its substrate (Qr) also increases in the presence of pyr-uvate and other a—keto acids (Millar et al 1996, Hoefnagel et al 1997) As a result, in the presence of the potent activator pyruvate the alternative path shows significant activity even when less than 30% of ubiquinone is in its reduced state, when the cyto-chrome pathway is not fully saturated (Fig 7) In intact tissues pyruvate levels appear to be suffi-ciently high to fully activate the alternative oxidase That is, changes in the level of keto acids probably not play a regulatory role in vivo (Hoefnagel & Wiskich 1998, Millenaar et al 1998)

Whenever the alternative oxidase is in its higher activity state and active at low levels of Qr, there will

be competition for electrons between the two path-ways, both in vitro (Hoefnagel et al 1995, Ribas-Carb ´o et al 1995) and in vivo (Atkin et al 1995) Competition for electrons between the two path-ways is the rule, rather than an exceptional situa-tion, as was initially thought

Does competition for electrons between the two pathways really occur at the levels of Qr that are commonly found in vivo (about 55% reduced; Millar et al 1998)? In vitro studies with mitochon-dria isolated from tissues of which we know that the alternative path contributes to respiration can pro-vide the answer (Fig 8B) In the presence of succi-nate, but no ADP (state 4; Fig 4), most of Q is reduced Upon addition of ADP (state 3; Fig 4), Q becomes more oxidized, until ADP is depleted Acti-vation of the alternative oxidase by pyruvate oxi-dizes Q to a level similar to that found in vivo

FIGURE9 Root respiration, growth and the activity of

isolated mitochondria for young Glycine max (soybean) seedlings (Top) O2 consumption via the cytochrome

path (circles) and the alternative path (squares), and the relative growth rate (diamonds) (Bottom) Succi-nate-dependent O2consumption and Q-pool reduction

Blocking the cytochrome path leads to Q being more reduced again Since the alternative oxidase contri-butes substantially to root respiration at a Qrlevel of 55%, the activation mechanisms must operate Because Qr levels in vivo are similar to those in state 4, Fig 8B also suggests that mitochondrial electron transport in roots is probably restricted by ADP (Fig 5)

2.6.3 Mitochondrial NAD(P)H Dehydrogenases That Are Not Linked to Proton Extrusion

In addition to the alternative oxidase (Sect 2.3.2) and the uncoupling proteins (Sect 2.3.3), there are NAD(P)H dehydrogenases that allow electron transport without proton extrusion (Møller 2001, Rasmusson et al 2004; Fig 3) Addition of NO3to N-limited seedlings of Arabidopsis thaliana (thale cress) decreases the transcript abundance of NAD(P)H dehydrogenase and alternative oxidase genes, while addition of NH4ỵdecreases the expres-sion of the same gene families Switching between NO3and NH4ỵin the absence of N stress leads to very similar results Corresponding changes in alternative respiratory pathway capacities are exhibited in seedlings supplied with either NO3 or NH4ỵas an N source and in mitochondria pur-ified from the seedlings (Escobar et al 2006) The parallel changes in both respiratory bypass path-ways suggests that the NAD(P) dehydrogenases play a similar role as the alternative oxidase (Sect 3)

3 The Ecophysiological Function of the Alternative Path

Why should plants produce and maintain a path-way that supports nonphosphorylating electron transport in mitochondria? Do they really differ fundamentally from animals in this respect, or animals have functional alternatives? Perhaps it is merely a relict or an ‘‘error’’ in the biochemical machinery that has not yet been eliminated by nat-ural selection On the other hand, there may be situations where respiration in the absence of ATP production could serve important physiological functions This Section discusses the merits of hypotheses put forward to explain the presence of the alternative path in higher plants Testing of these hypotheses will require the use of transgenics lack-ing alternative path activity, some of which are now available

3.1 Heat Production

An important consequence of the lack of coupling to ATP production in the alternative pathway is that the energy produced by oxidation is released as heat More than 200 years have passed since Lamarck described heat production in Arum italicum (Italian arum) and more than 70 years since thermo-genesiswas linked to cyanide-resistant respiration (Laties 1998) Thermogenesis has been reported for species in the Annonaceae, Araceae, Arecaceae, Aristolochiaceae, Cycadaceae, Nymphaeaceae, Winteraceae, Illiciaceae, Magnoliaceae, Rafflesia-ceae, and Nelumbonaceae (Seymour, 2001) This heat production is ecologically important in, e.g., Aymplocarpus renifoliusm (Asian skunk cabbage), which blooms in early spring when effective polli-nators are inactive (Sect 3.3.5 of Chapter on life cycles) During the female flowering phase, the spa-dices produce heat 24 hours per day, until the begin-ning of the male phase The spadices are visited by small numbers of invertebrate pollinators through-out the flowering season, attracted by the stench of aminesthat are volatilized by the elevated spadix temperature (Uemura et al 1993) During heat pro-duction the respiration of the spadix is largely cya-nide-resistant If the alternative pathway is indeed responsible for a major fraction of the spadix respiration, then this would contribute to the heat production, as the lack of proton extrusion coupled to electron flow allows a large fraction of the energy in the substrate to be released as heat This regulated thermogenic activity in inflorescences is function-ally analogous, but differs in biochemical mechan-ism, to the uncoupled respiration that occurs in thermogenic tissues (brown fat) of some mammals under cold conditions

Heat production also occurs in the flowers of sev-eral South American Annona species, Victoria amazo-nica(Amazon water lily) and Nelumbo nucifera (sacred lotus), clearly linked to activity of the alternative path (Fig 10) These flowers regulate their temperature with remarkable precision (Seymour et al 1998) When the air temperature varies between 10 and 308C, the flowers remain between 30 and 358C The stable temperature is a consequence of increasing respiration rates in proportion to decreasing tem-peratures Such a phenomenon of thermoregulation in plants is known for only a few species, e.g., Philo-dendron selloum(heart-leaf philodendron), Symplocar-pus foetidus(skunk cabbage) (Knutson 1974, Seymour 2001) It has been suggested that the heat produc-tion in lotus is an energetic reward for pollinating beetles These are trapped overnight, when they

feed and copulate, and then carry the pollen away (Seymour & Schultze-Motel 1996)

Can the alternative oxidase also play a significant role in increasing the temperature of leaves, for exam-ple during exposure to low temperature? There is indeed some evidence for increased heat production (7—22% increase) in low-temperature resistant plants (Moynihan et al 1995) It can readily be calculated, however, using an approach outlined in Chapter 4A on the plant’s energy balance, that such an increase in heat production cannot lead to a significant tempera-ture rise in leaves (less that 0.18C), and hence is

unlikely to play a role in any cold-resistance mechan-ism To explain the contribution of the alternative path in respiration of nonthermogenic organs other ecophysiological roles must be invoked

3.2 Can We Really Measure the Activity of the Alternative Path?

Does the alternative path also play a role in the respiration of ‘‘ordinary’’ tissues, such as roots and leaves? The application of specific inhibitors of the

FIGURE10 (Top)

Tempera-ture of the receptacle (Tr)

and ambient air (Ta) and

(Middle) rates of O2

con-sumption throughout the thermogenic phase in Nelu-mbo nucifera (sacred lotus) O2 consumption is

con-verted to heat production assuming 21.1 J ml-1of O

2

Shaded areas indicate the night period (Seymour & Schultze-Motel 1996) Rep-rinted with permission from Nature copyright 1996 MacMillan Magazines Ltd (Bottom) Total respiratory flux and fluxes through the alternative and cyto-chrome pathways, in lotus receptacle tissues as a function of the difference between receptacle tem-perature and temtem-perature of an adjacent nonheating receptacle Partitioning of electron transport between the two respiratory path-ways was determined on the basis of 18O-isotope

alternative path suggests that the alternative path does contribute to the respiration of roots and leaves of at least some species (Tables and 5) The decline in respiration, however, upon addition of an inhibi-tor of the alternative path tends to underestimate the actual activity of the alternative path If the two pathways compete for electrons, then the inhibition is less than the activity of the alternative path (Table 5) Thus, any observed inhibition of respira-tion following the addirespira-tion of an alternative path-way inhibitor indicates that some alternative pathway activity was present prior to inhibition, but provides no quantitative estimate of its activity (Day et al 1996)

Stable isotopescan be used to estimate alterna-tive path activity without the complications caused by use of inhibitors, because the alternative oxidase and cytochrome oxidase discriminate to a different extent against the heavy isotope of O2(Box 2B.1) The discrimination technique shows that the alter-native pathway may account for over 40% of all respiration The role of the alternative path in roots and leaves cannot be that of heat production What might be its role in these tissues?

3.3 The Alternative Path as an Energy Overflow

The activity of the alternative path might incre-ase when the production of organic acids is not matched by their oxidation, so that they accumu-late This observation led to the ‘‘energy overflow hypothesis’’ (Lambers 1982) It states that respira-tion via the alternative path only proceeds in the presence of high concentrations of respiratory sub-strate It considers the alternative path as a coarse control of carbohydrate metabolism, but not as an alternative to the finer control by adenylates (Sects 2.1 and 2.2)

The continuous employment of the alternative oxidase under normal ‘‘nonstress’’ conditions may ensure a rate of carbon delivery to the root that enables the plant to cope with ‘‘stress’’ According to the energy overflow hypothesis, if the carbon demand of a tissue suddenly increases, there is suf-ficient carbon transport to the tissue to meet these demands, if respiration were to switch entirely to supporting ATP synthesis For example, a decrease in soil water potential increases the roots’ carbon demand for synthesis of compatible solutes for osmotic adjustment Similarly, attack by parasites TABLE A comparison of the KCN resistance of

respiration of intact tissues of a number of species and of O2uptake by mitochondria isolated from these tissues.*

Cyanide-resistance (%)

Species Tissue

Whole

tissue Mitochondria

Gossypium hirsutum Roots 36 22 Phaseolus vulgaris Roots 61 41 Spinacia oleracea Roots 40 34 Triticum aestivum Roots 38 35

Zea mays Roots 47 32

Pisum sativum Leaves 39 30 Spinacia oleracea Leaves 40 27

Source: Lambers et al (1983)

*The percentage KCN resistance of intact tissue respiration

was calculated from the rate measured in the presence of 0.2 mM KCN and that measured in the presence of 0.1 mM FCCP, an uncoupler of the oxidative phosphorylation; this was done to obtain a rate of electron transfer through the cytochrome path closer to the state rate (Fig 4) KCN-resistance of isolated mitochondria was calculated from the rate in the presence and absence of 0.2 mM KCN Mitochon-drial substrates were 10 mM malate plus 10 mM succinate and a saturating amount of ADP KCN-resistant O2uptake

by isolated mitochondria was fully inhibited by inhibitors of the alternative path; in the presence of both KCN and SHAM approximately 10% of the control respiration proceeded in some of the tissues (‘‘residual respiration’’)

TABLE5 KCN-resistance, expressing the total respira-tory electron flow through the alternative path under the conditions of measurement, and SHAM-inhibi-tion of root respiraSHAM-inhibi-tion.*

Species KCN resistance SHAM inhibition

Carex diandra 66 29

Festuca ovina 53

Hordeum distichum 34

Pisum sativum 40 11

Phaseolus vulgaris 57 Plantago lanceolata 53 45

Poa alpina 41

Poa costiniana 61

Source: Atkin et al (1995)

*Values are expressed in percentage of the control rate of

respiration KCN and SHAM (salicylhydroxamic acid) are specific inhibitors of the cytochrome path and the alterna-tive path, respecalterna-tively Only if the cytochrome path is satu-rated, SHAM inhibition would equal the activity of the alternative path Since the cytochrome path is rarely satu-rated, SHAM-inhibition is usually less than the activity of the alternative path; in fact its activity may be as high as the KCN-resistant component of root respiration Because the two pathways generally compete for electrons, inhibitors cannot provide information on the actual activity of the two pathways in root respiration

Box 2B.1

Measuring Oxygen-Isotope Fractionation in Respiration

Plants have a cyanide-insensitive respiratory pathway in addition to the cytochrome pathway (Sect 2.3) Unlike the cytochrome pathway, the transport of electrons from ubiquinol to O2 through the alternative path is not linked to pro-ton extrusion, and therefore not coupled to energy conservation The alternative oxidase and cytochrome oxidase discriminate to a differ-ent extdiffer-ent against the heavy isotope of oxygen (18O) when reducing O2to produce water (Guy et al 1989) This allows calculation of the parti-tioning of electron flow between the two path-ways in the absence of added inhibitors, also in intact tissues For many years, studies of electron partitioning between the two respiratory path-ways were performed using specific inhibitors of the two pathways [e.g., cyanide for the cyto-chrome path, and SHAM (salicylhydroxamic acid) for the alternative path] It was thought that electrons were only available to the alterna-tive pathway when the cytochrome pathway was either saturated or inhibited; however, we now know that both pathways compete for electrons (Sect 2.6.1) The only reliable technique to study electron partitioning between the cytochrome and alternative pathway is by using oxygen-iso-tope fractionation (Day et al 1996) Although the methodology employed has changed dramati-cally in the last decade, the theoretical basis of the oxygen-isotope fractionation technique remains that described by Guy et al (1989)

The origin of the oxygen-fractionation metho-dology can be found in Bigeleisen & Wolfsberg (1959) and Mariotti et al (1981) Oxygen-isotope fractionation is measured by examining the isotope fractionation of the substrate O2as it is consumed in a closed, leak-tight cuvette The energy needed to break the oxygen-oxygen bond of a molecule con-taining18O is greater than that to break the mole-cule16O ¼ O16 Therefore, both terminal oxidases of the plant mitochondrial electron-transport chain react preferentially with 32O2, but they produce different isotope effects (Hoefs 1987) This allows determining the relative flux through each terminal oxidase If  is the ratio of the rate of the reaction with18O to that with16O, then:

Rp¼ R (1)

where Rpis the18O/16O ratio of the product (H2O), and R is that of the substrate (O2) Since  generally differs from unity by only a few per-cent, fractionation is usually given by D, where

D ẳ  ị  1000 (2)

and the units of D are parts per mil (%) D is generally obtained directly from Equation (1) by measurements of the isotope ratio of the sub-strate and product, but since the product of both mitochondrial oxidases is H2O, which is either the solvent for these reactions (liquid phase) or very difficult to obtain (gas phase), this is not feasible in this case Instead, changes in the iso-tope ratio of the O2 in the substrate pool are measured (Fig 1) If there is any isotopic fractio-nation during respiration, the oxygen-isotope ratio (R) of the remaining O2 increases as the reaction proceeds The respiratory isotope frac-tionation can be obtained by measuring R, and the fraction of molecular O2remaining at differ-ent times during the course of the reaction

Therefore, if we define the following terms:

Ro¼ initial18O/16O R ¼18O/16O at time t

f ¼ fraction of remaining oxygen at time t : f ẳ ẵo2=ẵo20

then the change in R through time is:

R=t ẳẵ 16

o18o=t 18o16o=t

16oị2 (3)

Since

18o=t ẳ R16o=tị (4)

we obtain:

R=R ẳ 16o=16o1  ị (5)

which, upon integration, yields

ln R=R0ẳ ln16o=16o0ð1  Þ (6)

Since only 0.4% of the O2 contains18O, the ratio16O/16Oois a good approximation of [O2]/ [O2o] (f), and hence we may write

and pathogens may suddenly increase carbon demands for tissue repair and the mobilization of plant defenses The alternative oxidase activity may also prevent the production of superoxide and/or

hydrogen peroxide under conditions where electron transport through the cytochrome path is impaired (e.g., due to low temperature or desiccation injury) This is partly due to a reaction of ubisemiquinone

Box 2B.1 Continued

D ẳlnR=R0ị=  ln f (7)

and D can be determined by the slope of the linear regression of a plot of ln R/Ro vs —ln f, without forcing this line through the origin (Henry et al 1999) The standard error (SE) of the slope is determined as

SE ẳD1  r 2ị1=2

rðn 2Þ1=2 (8)

and indicates the precision of the measurement of isotopic fractionation (D) This error should be less than 0.4%, because the fractionation differ-ential between the cytochrome pathway (18—20%) and the alternative pathway (24—31%) is between 6% and 12%, for roots and green tissues, respectively (Robinson et al 1995) In most cases, accurate determinations of D can be achieved with experiments comprising six mea-surements, providing the r2of the linear regres-sion is 0.995 or higher (Ribas-Carb ´o et al 1995, Henry et al 1999) Because it is common practice in the plant literature to express isotope

fractionation in ‘‘’’ notation, the fractionation factors, D, are converted to :

ẳ D

1  D=1000ị (9)

The partitioning between the cytochrome and the alternative respiratory pathways (a) is (Ribas-Carb ´o et al (1997):

a¼

n  c a  c

where n is the oxygen-isotope fractionation measured in the absence of inhibitors, and c and a are the fractionation by the cytochrome and alternative pathway, respectively These ‘‘end points’’ for purely cytochrome or alterna-tive pathway respiration are established for each experimental system using inhibitors of the alter-native oxidase and cytochrome oxidase, respec-tively The cytochrome oxidase consistently gives a c between 18% and 20%, while a is more variable, with values ranging from 24 to 25% in roots and nongreen tissues, and 30—32% in cotyledons and green leaves (Ribas-Carb ´o et al 2005b)

FIGURE1 Diagram of an on-line oxygen-isotope

frac-tionation system, with a gas-tight syringe, a stainless steel cuvette, a liquid nitrogen trap to remove CO2

and H2O, a reference bellow, and a sample bellow

(Ribas-Carb ´o et al 2005b)

with molecular O2(Purvis & Shewfelt 1993, Møller 2001) Superoxide, like other reactive oxygen spe-cies (ROS), can cause severe metabolic distur-bances So far, the various interpretations of the physiological function of an ‘‘energy overflow’’ remain speculative

3.4 NADH Oxidation in the Presence of a High Energy Charge

If cells require a large amount of carbon skeletons (e.g., oxoglutarate or succinate) but not have a high demand for ATP, then the operation of the alternative path could prove useful in oxidizing the NADH that would otherwise accumulate; con-sidering the pool size of NADH, this would then stop respiration within minutes However, can we envisage such a situation in vivo? Whenever the rate of carbon skeleton production is high, there tends to be a great need for ATP to further metabolize and incorporate these skeletons When plants are infected by pathogenic microorganisms, however, they tend to produce phytoalexins (Sect of Chapter 9C on effects of microbial pathogens) This generates sub-stantial amounts of NAD(P)H without major ATP requirements, and hence might require engagement of the alternative path (Sect 4.8)

There are also other circumstances where the production of carbon skeletons does not entail a need for ATP Cluster roots of Hakea prostrata (harsh hakea) accumulate large amounts of carbox-ylates (e.g., citrate), which they subsequently release to mobilize sparingly available P in the rhizosphere (Sect 2.2.5 of Chapter on mineral nutrition) Dur-ing the phase of rapid carboxylate synthesis, the alternative path is up-regulated, presumably allow-ing re-oxidation of NADH that is produced durallow-ing citrate synthesis (Shane et al 2004)

There may also be a need for a nonphosphorylat-ing path to allow rapid oxidation of malate in plants exhibiting crassulacean acid metabolism (CAM plants) during the day (Sect 10.2 of Chapter 2A on photosynthesis) Unfortunately, there are no techni-ques available to assess alternative path activity in the light If measurements are made in the dark, however, during the normal light period, then malate decarboxylation in CAM plants is indeed associated with increased engagement of the alter-native path (Table 6) Malate decarboxylation, how-ever, naturally occurs in the light (Sect 10.2 of Chapter 2A on photosynthesis) It therefore remains to be confirmed that the alternative path plays a vital role in CAM

3.5 NADH Oxidation to Oxidize Excess Redox Equivalents from the Chloroplast

In illuminated leaves, mitochondria are thought to play a role in optimizing photosynthesis Inhibition of either the cytochrome or the alternative path, using specific inhibitors (Sect 2.3.3), reduces photo-synthetic O2 evolution and the redox state of the photosynthetic electron transport chain in Vicia faba(broad bean) leaves under various light inten-sities Under saturating photosynthetic photon flux density, inhibition of either pathway causes a decrease in the steady-state levels of the photosyn-thetic O2 evolution rate and the PSII quantum yield Obviously, both two respiratory pathways are essential for maintenance of high photosyn-thetic rates at saturating light At low light inten-sity, however, only inhibition of the alternative path lowers the photosynthetic rate This suggests that inhibition of the alternative path causes over-reduction of the photosynthetic electron transport chain, even at low light levels

It has been suggested that an important function of the alternative oxidase is to prevent chloroplast over-reduction through efficient dissipation of excess reducing equivalents (Noguchi et al 2005) This hypothesis was tested using Arabidopsis thaliana (thale cress) mutants defective in cyclic electron flow around PSI, in which the reducing equivalents accumulate in the chloroplast stroma due to an unbalanced ATP/NADPH production ratio These mutants show enhanced activities of the enzymes needed to export the reducing equivalents from the TABLE Respiration, oxygen-isotope discrimination and partitioning of electrons to the cytochrome and the alternative pathway in leaves of Kalanchoe daigremontiana

Parameter Acidification De-acidification

Respiration 1.8 2.6

mmol O2m2s1

Discrimination 22.4 25.0 o/oo

Cytochrome path 1.3 1.4 mmol O2m2s1

Alternative path 0.5 1.2 mmol O2m2s1

Source: Robinson et al 1992

chloroplasts Interestingly, the amounts of alterna-tive oxidase protein and cyanide-resistant respira-tion in the mutants are also higher than those in the wild type After high-light treatment, the alternative oxidase, even in the wild type, is up-regulated con-comitant with the accumulation of reducing equiva-lents in the chloroplasts and an increase in the activities of enzymes needed to export reducing equivalents These results indicate that the alterna-tive oxidase can dissipate excess reducing equiva-lents that are exported from the chloroplasts, and that it plays a role in photosynthesis (Yoshida et al 2007)

3.6 Continuation of Respiration When the Activity of the Cytochrome Path Is Restricted

Naturally occurring inhibitors of the cytochrome path (e.g., cyanide, sulfide, carbon dioxide, and nitric oxide) may reach such high concentrations in the tissue that respiration via the cytochrome path is partially or fully inhibited (Palet et al 1991, Millar & Day 1997) Similarly, mutants of that lack complex I (Karpova et al 2002) and hence must use the non-phosphorylating bypass, produce less ATP than the wild type, if respiring at the same rate Under these circumstances the alternative pathway may be important in providing energy, even though it yields only a third as much ATP as the cytochrome path This has indeed been shown to be the case for a Nicotiana sylvestris(flowering tobacco) mutant that lacks complex I, using the oxygen-isotope fractiona-tion technique (Box 2B.1; Vidal et al 2007)

Dry seeds, including those of Cucumis sativus (cucumber), Hordeum vulgare (barley), Oryza sativa (rice), and Xanthium pennsylvanicum (cocklebur) con-tain cyanogenic compounds, such as cyanohydrin, cyanogenic glycosides, and cyanogenic lipids Such compounds liberate free HCN after hydrolysis during imbibition Upon imbibition and triggered by ethy-lene, seeds containing these cyanogenic compounds produce a mitochondrial b-cyano-alanine synthase that detoxifies HCN (Hagesawa et al 1995) Despite this detoxifying mechanism, some HCN is likely to be present in the mitochondria of germinating seeds, and hence there is a need for a cyanide-resistant path

Some plants produce sulfide (e.g., species belong-ing to the Cucurbitaceae) (Rennenberg & Filner 1983) Sulfide is also produced by anaerobic sulfate-reducing microorganisms It may occur in high concentrations in the phyllosphere of aquatic plants or the rhizo-sphere of flooded plants In such flooded soils, carbon dioxidelevels also increase Since both sulfide and

high concentration of carbon dioxide inhibit the cyto-chrome path (Palet et al 1991), there may be a need for the alternative path under these conditions also

When the activity of the cytochrome path is restricted by low temperature, the alternative path might also increase in activity to provide energy needed for metabolism In fact, sustained exposure to low temperature enhances the amount of alterna-tive oxidase in mitochondria of Zea mays (corn) (Stewart et al 1990) and Nicotiana tabacum (tobacco) (Vanlerberghe & McIntosh 1992) Such an induction also occurs when the activity of the cytochrome path is restricted in other ways [e.g., by application of inhibitors of mitochondrial protein synthesis (Day et al 1995), or of inhibitors of the cytochrome path (Wagner et al 1992)] Interestingly, only those inhi-bitors of the cytochrome path that enhance super-oxide production lead to induction of the alternative oxidase, suggesting that the prevention of damage by reactive oxygen species is a particularly impor-tant role of the alternative path Moreover, super-oxide itself can also induce expression of the alternative oxidase This has led to the suggestion that reactive oxygen species, including H2O2, are part of the signal(s) communicating cytochrome path restriction in the mitochondria to the nucleus, thus inducing alternative oxidase synthesis (Rhoads et al 2006) The key question is, of course, if enhanced expression of the alternative oxidase leads to greater activity of the alternative path In Vigna radiata (mung bean) this appears to be the case, but such a response is not found in Glycine max (soybean) (Gonza`lez-Meler et al 1999)

In the absence of an alternative oxidase, inhibi-tion or restricinhibi-tion of the activity of the cytochrome path would inexorably lead to the accumulation of fermentation products, as found in transgenic plants lacking the alternative oxidase (Vanlerberghe et al 1995) In addition, it might cause the ubiqui-none pool to become highly reduced which might lead to the formation of reactive oxygen species and concomitant damage to the cell (Purvis & Shewfelt 1993, Møller 2001) Further work with transgenics lacking the alternative path is an essential avenue of future research on the ecophysiological role of the alternative path in plant functioning

3.7 A Summary of the Various

Ecophysiological Roles of the Alternative Oxidase

The alternative oxidase is widespread and can serve a wide variety of physiological functions, ranging from providing ATP when the cytochrome pathway is

restricted (which can occur under a wide variety of circumstances) to acting as an overflow to balancing the physiological rates of a range of processes (e.g., organic acid synthesis and ATP production) to prevent metabolism from getting severely unbalanced In higher plants, the alternative pathway is just as much entrained in all aspects of metabolism as is the cytochrome path In addition to the alternative path, plants have a bypass of complex I and uncoupling proteins The reason why most animals not have an alternative respiratory path is probably that they entirely depend on uncou-pling proteins In addition, animals may have less need to balance different metabolic functions

4 Environmental Effects on Respiratory Processes

4.1 Flooded, Hypoxic, and Anoxic Soils

Plants growing in flooded soil are exposed to hypoxic (low-O2) or anoxic (no-O2) conditions in the root environment, and experience a number of conditions, including an insufficient supply of O2 and accumulation of CO2(Sect 4.7), and changes in plant water relations (Sect of Chapter on plant water relations)

4.1.1 Inhibition of Aerobic Root Respiration

The most immediate effect of soil flooding on plants is a decline in the O2concentration in the soil In water-saturated soils the air that is normally present in the soil pores is almost completely replaced by water The diffusion of gases in water is approxi-mately 10000 times slower than in air In addition, the concentration of O2in water is much less than that in air (at 258C approximately 0.25 mmol O2 dissolves per liter of water, whereas air contains approximately 10 mmol) The O2supply from the soil, therefore, decreases to the extent that aerobic

root respiration, and hence ATP production, is restricted Under these conditions the synthesis of RNA and proteins is strongly suppressed, but that of specific m-RNAs and anaerobic polypeptides is induced Among these ‘‘anaerobic polypeptides’’ is the fermentative enzyme alcohol dehydrogenase (Andrews et al 1993)

4.1.2 Fermentation

When insufficient O2reaches the site of respiration, such as in seeds germinating under water and sub-merged rhizomes, ATP may be produced through fermentative processes These tissues generate energy in glycolysis, producing ethanol, and some-times lactate Lactate tends to be the product of fer-mentation immediately after the cells are deprived of O2 Lactate accumulation decreases the pH in the cytosol (Sect 4.1.3), which inhibits lactate dehydro-genase and activates the first enzyme of ethanol fer-mentation: pyruvate decarboxylase When lactate accumulation does not stop, cytosolic acidosis may lead to cell death (Rivoal & Hanson 1994)

It was initially believed that root metabolism cannot continue in flooded conditions, due to the production of toxic levels of ethanol Ethanol, how-ever, does not really inhibit plant growth until con-centrations are reached that far exceed those found in flooded plants (Table 7), and hence ethanol plays only a minor role in flooding injury to roots and shoots (Jackson et al 1982) As long as there is no accumulation of acetaldehyde, which is the pro-duct of pyruvate decarboxylase and the substrate for alcohol dehydrogenase, which reduces acetal-dehyde to ethanol, alcoholic fermentation is unli-kely to cause plant injuries If acetaldehyde does accumulate, however, for example upon re-aeration, then this may cause injury, because acet-aldehyde is a potent toxin, giving rise to the forma-tion of reactive oxygen species (Blokhina et al 2003) It is the low potential for ATP production and its metabolic consequences, rather than the

TABLE7 The effect of supplying ethanol in aerobic and anaerobic nutrient solutions to the roots of Pisum sativum (garden pea) at a concentration close to that found in flooded soil (i.e., 3.9 mM) or greater than that

Aerobic control Aerobic ỵ ethanol Anaerobic control Anaerobic ỵ ethanol

Ethanol in xylem sap (mM) 37 540 90 970

Stem extension (mm) 118 108 94 74

Final fresh mass (g)

shoot 11.9 11.9 10.7 11.4

roots 7.8 9.7 5.7 6.1

toxicity of the products of fermentative metabolism that constrain the functioning of plants under anoxia (Sect 4.1.3)

Continued fermentation requires the mobiliza-tion of a large amount of reserves, such as starch Seeds of most species fail to germinate under anoxia, but those of Oryza sativa (rice) are an excep-tion (Perata & Alpi 1993) In contrast to cereals like Triticum aestivum(wheat) and Hordeum vulgare (bar-ley), rice seeds produce -amylase and sucrose-metabolizing enzymes under anoxia; these enzymes allows the degradation and further metabolism of starch, and therefore sustain a rapid fermentative metabolism (Perata et al 1996)

The energetic efficiency of ethanol formation is low, producing only two molecules of ATP in gly-colysis (‘‘substrate phosphorylation’’) per molecule of glucose This is considerably less than that of aerobic respiration, which produces around 36 molecules of ATP per molecule of glucose, if the most efficient mitochondrial electron-transport pathways are used and not taking into account the costs for transport of metabolites across the inner mitochondrial membrane (Sects 2.2 and 2.3) Moreover, a large fraction of the lactate may be secreted into the rhizosphere [e.g., in some Limo-nium (statice) species] Although such secretion

prevents acidification of the cytosol, it also repre-sents a substantial carbon loss to the plant (Rivoal & Hanson 1993)

4.1.3 Cytosolic Acidosis

A secondary effect of the decline in root respiration and ATP production in the absence of O2 is a decrease in the pH of the cytosol (cytosolic acido-sis), due in part to accumulation of organic acids in fermentation and the TCA cycle Moreover, in the absence of O2as a terminal electron acceptor, ATP production decreases, so there is less energy avail-able to maintain ion gradients within the cell Acid-ification of the cytosol reduces the activity of many cytosolic enzymes, whose pH optimum is around and hence severely disturbs the cell’s metabolism, so that protons leak from the vacuole to the cytosol Cytosolic acidosis also reduces the activity of aqua-porins (Sect 5.2 of Chapter on plant water rela-tions) The extent of this cytosolic acidification is less in the presence of NO3(Fig 11) NO3reduction leads to the formation of hydroxyl ions (Sect 2.2.6.1 of Chapter on mineral nutrition), which partly neutralize the protons and prevent severe acidosis Moreover, NO3reduction requires the oxidation of NADH, producing NAD This allows the continued

FIGURE11 The effect of hypoxia on root tips of Zea

mays (maize), in the presence (triangles) and absence (circles) of nitrate (A) The effect on the pH of the cytosol, as measured in experiments using31P-NMR spectroscopy; (B) the increase in fresh mass during 48 hours in air, after the indicated period of hypoxia The location of the inorganic phosphate (Pi) peaks in

an NMR spectrum depends on the ‘‘environment’’ of the molecule (e.g., pH) (Fig in this chapter) NMR spectroscopy can therefore be used to determine the peak wavelength at which Piabsorbs the magnetic

radiation and hence the pH in the cytosol as well as in the vacuole (after Roberts et al 1985) Copyright American Society of Plant Biologists

oxidation of organic acids in the TCA cycle, thus preventing their accumulation and associated drop in pH

4.1.4 Avoiding Hypoxia: Aerenchyma Formation

In wetland plants, including crop species such as Oryza sativa(rice), mechanisms have evolved to pre-vent the problems associated with flooded soils The most important adaptation to flooded soils is the development of a functional aerenchyma, a contin-uous system of air spaces in the plant that allows diffusion of O2from the shoot or the air to the roots (Jackson & Armstrong 1999) Aerenchyma avoids inhibition of respiration due to lack of O2which is inevitable for plants that are not adapted to wet soils (Colmer 2003b) In many species, other special struc-tures allow the diffusion of O2from the air into the plant: the pneumatophores of mangroves, lenticels in the bark of many wetland trees, and, possibly, the knee roots of Taxodium distichum (bald cypress) The mechanisms that maintain the intercellular spaces filled with gas rather than water are not fully under-stood Inward radial gradients in water potential created by transpiration in combination with water-impermeable apoplastic barriers such as the exodermis may offer an explanation (Jackson & Armstrong 1999)

Because there is a gradient in partial pressure within the aerenchyma, O2will move by diffusion to the roots In aquatic plants, however, like Nuphar lutea(yellow water lily) and Nelumbo nucifera (sacred lotus) there is also a pressurized flow-through system, which forces O2 from young emergent leaves to the roots and rhizomes buried in the anae-robic sediment (Dacey 1980, 1987) Such a mass flow requires a difference in atmospheric pressure between leaves and roots The diurnal pattern of the mass flow of air to the roots suggests that the energy to generate the pressure comes from the sun; however, it is not the photosynthetically active com-ponent of radiation, but the long-wave region (heat), which increases the atmospheric pressure inside young leaves by as much as 300 Pa How can these young leaves draw in air against a pressure gradi-ent? To understand this we have to realize that the atmosphere inside the leaf is saturated with water vapor and that movement of gases occurs by diffu-sion, along a gradient in partial pressure, and by mass flow, depending on the porosity of the path-way The porosity of the young emergent leaves is such that gas flux by diffusion (i.e., down a concen-tration gradient) is more important than a mass flux

due to a difference in atmospheric pressure The concentration gradient is due to the evaporation from the cells inside the leaf, which dilutes the other gases in the intercellular spaces, thus creating a gradient allowing diffusion between the atmo-sphere and the intercellular spaces The slightly higher atmospheric pressure inside young leaves forces air, which has been enriched in O2by photo-synthesis, to move along a pressure gradient from young leaves to roots and rhizomes Some of the air from roots and rhizomes, which is enriched with CO2from respiration, is then forced to older leaves Isotope studies show that much of this CO2is sub-sequently assimilated in photosynthesis The reason that only young leaves show this internal ventilation is the higher porosity of the older leaves which does not allow them to draw in more air through diffu-sion than is lost via mass flow The quantity of air flow through a single petiole is enormous: as much as 22 liters per day, with peak values as high as 60 ml per minute and rates of 50 cm per minute The transport of O2 from the shoot by convective gas flow is also likely to contribute to the flow of O2to roots of other species growing in an anaerobic soil (Armstrong et al 1997) Pressurized flow of O2plays a role in the O2supply to the roots and rhizosphere of many emergent macrophytes The vital element is that a compartment exists surrounded by walls with sufficiently small pores to allow diffusion to occur at greater rates than mass flow (Colmer 2003a,b)

Aerenchymatous plants often transport more O2 to the roots than is consumed by root respiration The outward diffusion of O2into the rhizosphere implies a loss of O2 for root respiration Plants adapted to flooded conditions, e.g., Oryza sativa (rice), Phragmitis australis (common reed) and Glyceria maxima (reed mannagrass) develop a flooding-induced O2barrierin basal root zones, thus reducing radial O2 loss (Colmer 2003a, Soukup et al 2007) On the other hand, outward diffusion of O2also allows the oxidation of poten-tially harmful compounds (Colmer 2003b) This can readily be seen when excavating a plant from a reduced substrate The bulk substrate itself is black, due to the presence of FeS, but the soil in the immediate vicinity of the roots of such a plant will be brown or red, indicating the presence of oxidized iron (Fe3+, ‘‘rust’’), which is less soluble than the reduced Fe2+

uptake is strongly affected by root diameter and surface area, a likely cost associated with aerench-yma is a reduced rate of nutrient uptake per unit root biomass The basal O2barrier, which involves both quantitative and qualitative differences in suberincomposition and distribution within exo-dermal cell walls (Soukup et al 2007) probably also decreases the roots’ capacity for nutrient and water uptake

Aerenchyma also serves as a conduit of soil gases to the atmosphere, including methane, ethylene, and carbon dioxide Methane (CH4) is a bacterial product commonly produced in anaerobic soils In rice paddies and natural wetlands most CH4 is transported to the atmosphere through plant aer-enchyma Experimental removal of sedges from wetland substantially reduces CH4flux and causes CH4to accumulate in soils (Fig 12) CH4production and transport to the atmosphere is a topic of current concern, because CH4 is a ‘‘greenhouse gas’’ that absorbs infrared radiation 20 times more effectively than does CO2 Recent increases in atmospheric CH4 have contributed approximately 20% of the warm-ing potential of the atmosphere that has caused recent global warming (Ramaswamy et al 2001) The expansion of rice agriculture and associated CH4transport via aerenchyma from the soil to the atmosphere is an important contributor to atmo-spheric CH4 There is no firm evidence that plants themselves generate significant amounts of CH4, and suggestions in the literature that planting trees might contribute to a major extent to global warm-ing due to their aerobic production of CH4have not been substantiated (Dueck et al 2007, Kirschbaum et al 2007)

4.2 Salinity and Water Stress

Sudden exposure of sensitive plants to salinity or water stress often enhances their respiration For example, the root respiration of Hordeum vulgare (barley) increases upon exposure to 10 mM NaCl (Bloom & Epstein 1984) This may either reflect an increased demand for respiratory energy or an increased activity of the alternative path, when car-bon use for growth is decreased more than carcar-bon gain in photosynthesis (Sect 5.3 of Chapter on growth and allocation) Long-term exposure of sen-sitive plants to salinity or desiccation gradually decreases respiration as part of the general decline in carbon assimilation and overall metabolism asso-ciated with slow growth under these conditions (Galme´s et al 2007; Sect 5.3 of Chapter on growth and allocation) Generally, specific rates of leaf respiration at 258C are highest in plants growing in hot, dry habitats, reflecting acclimation and/or adaptation to such habitats (Wright et al 2006) Additional declines in root respiration of Triticum aestivum (wheat) plants upon exposure to dry soil may reflect a specific decline in the alternative path The decline correlates with the accumulation of osmotic solutes, reducing the availability of sugars and hence providing less ‘‘grist for the mill’’ of the alternative path

Leaves also show a decline in respiration, as leaf water potential declines The decline is most likely associated with a decrease in the energy requirement for growth or the export of photoassimilates In Gly-cine max(soybean) net photosynthesis decreases by 40% under mild and by 70% under severe water stress, whereas the total respiratory O2uptake is not significantly different at any water-stress level How-ever, severe water stress causes a significant shift of electrons from the cytochrome to the alternative pathway The electron partitioning through the alter-native pathway increases from about 11% under well watered or mild water-stress conditions to near 40% under severe water stress (Fig 13) Consequently, the calculated rate of mitochondrial ATP synthesis decreases by 32% under severe water stress (Ribas-Carb ´o et al 2005a)

Species differ in their respiratory response to water stress, primarily due to differences in sensi-tivity of growth to desiccation When salt-adapted plants are exposed to mild salinity stress, they accu-mulate compatible solutes, such as sorbitol (Sect of Chapter on plant water relations) Accumula-tion of these sugar alcohols requires glucose as a substrate but does not directly affect the concentra-tion of carbohydrates or interfere with growth

FIGURE12 Methane flux and soil methane concentration

in a tundra wetland in which sedges are present (control) or have been experimentally removed (data from Torn & Chapin 1993)

Studies of root respiration, using an inhibitor of the alternative path, suggested that sorbitol accumula-tion is associated with a reducaccumula-tion in activity of the nonphosphorylating alternative respiratory path-way However, further experimentation using the oxygen-isotope fractionation technique (Box 2B.1) is required to confirm this Interestingly, the amount of sugars that are ‘‘saved’’ by the decline in respira-tion is the same as that used as the substrate for the synthesis of sorbitol, suggesting that accumulation of compatible solutes by drought-adapted plants may have a minimal energetic cost (Lambers et al 1981)

Prolonged exposure of salinity-adapted species (halophytes) to salt concentrations sufficiently low not to affect their growth has no effect on the rate of root respiration This similarity in growth and respiratory pattern under saline and nonsaline con-ditions suggests that the respiratory costs of coping with mild salinity levels are negligible in salt-adapted species The respiratory costs of function-ing in a saline environment for adapted species that accumulate NaCl are also likely to be relatively small, because of the low respiratory costs of absorb-ing and compartmentalizabsorb-ing salt when grown in saline soils For salt-excluding glycophytes, how-ever, there may be a large respiratory cost associated with salt exclusion

4.3 Nutrient Supply

Root respiration generally increases when roots are suddenly exposed to increased ion concentrations in

their environment, a phenomenon known as salt respiration The stimulation of respiration is at least partly due to the increased demand for respiratory energy for ion transport The added respiration may also reflect a replacement of osmo-tically active sugars by inorganic ions, leaving a large amount of sugars to be respired via the alter-native path

When plants are grown at a low supply of N, their rate of root respiration is lower than that of plants well supplied with mineral nutrients (Atkinson et al 2007) This is expected because their rates of growth and ion uptake are greatly reduced (Fig 14) Rates of root respiration, however, per ion absorbed or per unit root biomass produced at a low NO3 sup-ply are relatively high, if we compare these rates with those of plants that grow and take up ions at a much higher rate This suggests that specific costsof growth (that is cost per unit biomass pro-duced), maintenance (cost per unit biomass to be maintained), or ion transport (cost per unit nutrient absorbed) must increase in plants grown at a limit-ing nutrient supply (Sect 5.2.4)

There is also a correlation between leaf respira-tionand leaf N concentration (Loveys et al 2003, Noguchi & Terashima 2006) Although the correla-tion between leaf respiracorrela-tion and leaf N concentra-tion tends to be general, irrespective of the natural habitat of the species (Tjoelker et al 1999, Reich et al 2006), environment-mediated changes in the rela-tionship between leaf respiration and leaf N can occur For example, in a comparison of 70 Australian perennial species, the slope of leaf respiration (on a dry mass basis) vs leaf N concentration is constant across sites, but there are differences in the intercept for sites differing in nutrient availability and rainfall (Wright et al 2001) The physiological basis for such a difference in intercept remains to be explored

4.4 Irradiance

The respiratory response of plants to light and assimilate supply depends strongly on time scale The immediate effect of low light is to reduce the carbohydrate status of the plant and, therefore, the supply of substrate available for respiration (Fig 15A) Interestingly, in the shade species Alocasia odora (Asian taro) addition of sucrose does not increase the rate of leaf respiration of plants transferred to the shade (Fig 15B), but addi-tion of an uncoupler does increase respiraaddi-tion to a major extent (Fig 15C) (Noguchi et al 2001a) This shows that respiration is controlled by energy

FIGURE13 Effect of different levels of water stress on

total respiration (Vt), the activities of the cytochrome

(vcyt), and alternative (valt) pathways and the

partition-ing through the alternative pathway (ta) (after

demand, rather than substrate supply (Sect 2.4, Fig 5), and that the energy demand is down-regu-lated in shade conditions In the leaves of Alocasia odora, the contribution of the alternative path is less than 10% of the total respiratory rate, irrespec-tive of growth irradiance For the sun species Spi-nacia oleracea (spinach) and Phaseolus vulgaris (common bean) grown at high light intensity, the contribution of the alternative path in the leaves is about 40% early in the night, but decreases drama-tically late in the night When spinach is grown at low light intensity, however, the contribution of the alternative path in the leaves declines The low activity of the alternative path in the leaves of the understory species Alocasia odora shows that the efficiency of ATP production (ADP:O ratio) of this species is high This may be especially important in shade environments In the leaves the of sun spe-cies, the ADP:O ratio changes depending on con-ditions (Noguchi et al 2001a)

To further investigate why the understory spe-cies Alocasia odora (Asian taro) consistently shows low alternative path activity, Noguchi et al (2005) grew Alocasia odora and Spinacia oleracea (spinach) plants under both high and low light intensities On a mitochondrial protein basis, Spinacia oleracea leaves show a higher capacity of the cytochrome pathway than Alocasia odora leaves Despite a low in vivo activity of the alternative path, Alocasia odora has a higher capacity of the alternative oxi-dase on a mitochondrial protein basis In the low-light environment, most of the alternative oxidase

protein in Alocasia odora leaves is in its inactive, oxidized dimerform (Sect 2.6.2), but it is conver-ted to its reduced, active form when plants are grown under high light (Fig 16) This shift may pre-vent over-reduction of the respiratory chain under photo-oxidative conditions

Roots and leaves that are subjected to an increased or decreased carbohydrate supply gra-dually acclimate over several hours by adjusting their respiratory capacity Upon transfer of Poa annua (annual meadow-grass) from high-light to low-light conditions, and at the same time from long-day to short-day conditions, the sugar con-centrationin the roots decreases by 90% Both the rate of root respiration and the in vitro cytochrome oxidasecapacity decrease by about 45%, relative to control values The absolute rate of O2uptake via the alternative pathway, as determined using the isotope fractionation technique (Box 2B.1), does not change, but the cytochrome pathway activity decreases Interestingly, there is no change in the concentration of the alternative oxidase protein or in the reduction state of the protein Also, there is no change in the reduction state of the ubiquinone pool These results show that neither the amount nor the activity of the alternative oxidase change under severe light deprivation (Millenaar et al 2000), suggesting an important role for this appar-ently wasteful pathway; this role is most likely avoiding production of reactive oxygen species, as discussed in Sect 3.3 The results also point to acclimation of respiration as a result of changes in

FIGURE14 (A) Rates of net inflow of nitrate of six grass

species grown at two nitrogen addition rates, allowing a near-maximum relative growth rate (open columns) or a RGR well below RGRmax(black columns) (B) Root

respiration of the same inherently fast- and slow-grow-ing grasses as shown in A, now compared at a range of nitrogen addition rates allowing a near-maximum rela-tive growth rate or a relarela-tive growth rate below RGRmax, the lowest RGR being 38 mg g1day1 Cd,

Carex diandra (lesser panicled sedge) (open circles); Cf, Carex flacca (blue sedge) (filled triangle); Bm, Briza media (quacking grass) (filled squares); BP, Brachypo-dium pinnatum (Tor grass) (filled circles); DG, Dactylis glomerata (cocksfoot) (open squares); Hl, Holcus lana-tus (common velvet grass) (open triangles) (Van der Werf et al 1992a) Copyright SPB Academic Publishing

gene expression Also, after pruning of the shoot to one leaf blade, both the soluble sugar concen-tration and the respiration of the seminal roots decrease These effects on respiration reflect the coarse control of the respiratory capacity upon pruning or sucrose feeding (Bingham & Farrar 1988, Williams & Farrar 1990) This illustrates the adjustment of the respiratory capacity to the root’s carbohydrate level

Changes in respiratory capacity induced by changes in carbohydrate status reflect acclimation of the respiratory machinery The protein pattern of the roots of pruned plants is affected within 24 h (McDonnel & Farrar 1992, Williams et al 1992) Glu-cose feeding to leaves enhances the activity of sev-eral glycolytic enzymes in these leaves, due to

regulation of gene expression by carbohydrate levels (Krapp & Stitt 1994) Clearly, the capacity to use carbohydrates in respiration is enhanced when the respiratory substrate supply increases, and declines with decreasing substrate supply The plant’s potential to adjust its respiratory capacity to environmental conditions is ecologically signifi-cant Individual plants acclimated to low light gen-erally have low leaf respiration rates Thus acclimation accentuates the short-term declines in respiration due to substrate depletion

As with acclimation, species that are adapted to low light generally exhibit lower respiration rates than high-light adapted species For example, the rainforests understory species of Alocasia odora (Asian taro) has lower rates of both photosynthesis

FIGURE15 Leaf respiration in the shade species

Alo-casia odora (Asian taro) as dependent on light avail-ability (A) Changes in the rate of O2uptake Effects

of (B) the addition of an uncoupler (FCCP) and (C) a respiratory substrate (sucrose) on the rate of O2

FIGURE16 (A) Immunoblots of the alternative oxidase

(AOX) in extracted membrane fractions isolated from Alocasia odora (Asian taro) leaves Extractions were made very rapidly, so as to maintain the activation state of AOX and determine its in vivo state Lane 1, a sample of leaves of plants grown at very low light inten-sities (VLL) treated in the presence of 50 mM DTT (dithiothreitol, which renders AOX in its reduced and active state, irrespective of its state in vivo) Lane 2, a sample of VLL leaves treated in the presence of mM diamide (which oxidizes and inactivates the AOX dimer, irrespective of its state in vivo) Lanes and 4, samples consisted of only VLL leaf membrane fractions; the immunoblots show that AOX was in its oxidized, inac-tive state in leaves of plants grown at very low light intensity Lanes and 6, samples consisted of only high-light grown (HL) leaf membrane fractions; these immunoblots show that AOX was in its reduced, active state in leaves of plants grown at high light intensity (B)

Immunoblots of AOX in rapidly extracted membrane fractions and/or mitochondria isolated from Alocasia odora leaves Lane 7, a sample consisted of only VLL leaf membrane fractions; AOX was in its oxidized, inac-tive state Lane 8, a sample of VLL leaf membrane frac-tions, added with a mitochondrial extract from HL leaves just before the extraction; Lane 9, mitochondrial sample isolated from HL leaves; during isolation some AOX is reduced and activated (Noguchi et al 2005) (C) Under very low light conditions, the alternative oxidase is in its inactive, oxidized form (left) It is converted to its reduced, active form (right) when plants are exposed to high-light conditions This shift may prevent over-reduc-tion of the respiratory chain under photo-oxidative con-ditions The structural model for AOX has been deduced from derived amino acid sequences and is reprinted with permission of the American Society of Plant Biolo-gists Photographs by K Noguchi Copyright Blackwell Science Ltd

and respiration than does the sun species Spina-cia oleracea (spinach), when the two species are compared under the same growth conditions (Table 8) The net daily carbon gain of the leaves (photosynthesis minus respiration) is rather simi-lar for the two species, when expressed as a proportion of photosynthesis Similarly, unders-tory species of Piper (pepper) have lower respira-tion rates than species from shaded and exposed habitats, when both are grown in the same envir-onment (Fredeen & Field 1991) Because rates of photosynthesis and respiration show parallel dif-ferences between sun and shade species (both lower in the shade species), differences in the carbon balance between sun and shade species probably reflect different patterns of biomass allocation rather than differences in photosynth-esis and respiration

Respiration rates tend to be higher in plants grown at higher light intensity Acclimation to higher levels of irradiance involves up-regulation of genes involved in the metabolism of carbohy-drates and in energy-requiring processes (coarse control) In the short term, respiration may respond to irradiance because this affects the availability of respiratory substrate (control by supply) Sudden exposure of shade plants to a high light intensity may require a change in activation state of the alter-native oxidase, associated with accumulation of reactive oxygen species (stress response)

Acclimation of respiration is relatively fast (hours to days), when compared with that of photo-synthesis (days to weeks) This is largely accounted for by the fact that some aspects of photosynthetic acclimation require the production of new leaves with a different structure, whereas acclimation of respiration requires only production of new proteins

4.5 Temperature

Respiration increases as a function of temperature, with the magnitude of increase depending on the temperature coefficient (Q10) of respiration This temperature effect on respiration is characteristic of most heterothermic organisms and is a logical consequence of the temperature sensitivity of the enzymatically catalyzed reactions involved in respiration The temperature stimulation of respira-tion also reflects the increased demand for energy to support the increased rates of biosynthesis, trans-port, and protein turnover that occur at high tem-peratures (Sect 5.2)

Temperature-mediated changes in plant respira-tion are an important component of the biosphere’s response to global climate change The Q10is often modeled to be (i.e., respiration doubles per 108C rise in temperature) However, upon longer-term exposure to a different temperature, the initial tem-perature effect of a Q10of may diminish, and the long-term Q10declines predictably with increasing temperature across diverse plant taxa and biomes (Fig 17A) This is due to thermal acclimation, i.e., the adjustment of respiration rates to compensate for a change in temperature The temperature dependence of Q10is linked to shifts in the control by maximum enzyme activity at low temperature and substrate limitations at high temperature (Fig 17B) In the long term, acclimation of respiration to temperature is common, reducing the temperature sensitivity of respiration to changes in thermal envir-onment Temperature acclimation results in a ten-dency toward homeostasis of respiration, such that warm-acclimated (temperate, lowland) and cold-acclimated alpine or high-arctic plants display simi-lar rates of respiration when measured at their TABLE8 The daily carbon budget (mmol g1day1) of the leaves of Spinacia oleracea (spinach), a sun species, and Alocasia odora (giant upright elephant ear), a shade species, when grown in different light environments.*

Irradiance

Photosynthesis Leaf respiration Net leaf carbon gain

Spinacia oleracea

Alocasia odora

Spinacia oleracea

Alocasia odora

Spinacia oleracea

Alocasia odora

500 26 nd 3.4 (13) nd 23 (87) nd

320 21 11 2.4 (12) 1.1 (10) 18 (88) 9.4 (90)

160 15 1.7 (11) 0.82 (9) 14 (89) 8.2 (91)

40 nd 4.5 nd 0.76 (17) nd 3.7 (83)

Source: Noguchi et al (1996), K Noguchi, pers comm

* Irradiance is expressed in mmol m2s1 Percentages of the photosynthetic carbon gains have been indicated in brackets;

respective growth temperatures; however, complete homeostasis is uncommon Acclimation can play an important role in weakening positive feedback through the warming-respiration-atmospheric CO2 concentration connection (Atkin & Tjoelker 2003) Acclimationof leaf respiration to temperature is lar-ger in conifers than in broad-leaved species (Tjoelker et al 1999); other than that, there are no major sys-tematic differences in the degree of acclimation among contrasting plant species (Loveys et al 2003) The mechanism of temperature acclimation of respiration is not yet fully understood At low mea-surement temperatures (e.g., 58C), respiratory flux is probably limited by the Vmax(Covey-Crump et al 2002) (lower panels in Fig 17) of the respiratory apparatus [i.e., glycolysis, the TCA cycle, and mito-chondrial electron transport (Sects 2.2 and 2.3)] At moderately high temperatures (e.g., 258C), respira-tory flux is less limited by enzymatic capacity because of increases in the Vmaxof enzymes in solu-ble and membrane-bound compartments; here, respiration is likely limited by substrate availability

and/or adenylates Increased leakiness of mem-branes at high temperatures may further contribute to substrate limitations The net result of tempera-ture-mediated shifts in control from capacity (at low temperatures) to substrate or adenylate limitation (at moderately high temperatures) (Fig 17) is that a rise in measurement temperature has less impact on respiratory flux at moderate-high temperatures than it does in the cold As a result, the calculated Q10is lower when calculated across a high measure-ment temperature range than at a range of low mea-surement temperatures To firmly establish if respiratory enzyme capacity limits respiratory flux in the cold, data are needed on the maximum poten-tial flux of the respiratory apparatus in intact tissues at low temperatures These can be obtained via mea-surements of respiration in isolated mitochondria, in the presence of saturating substrates and ADP (Fig 4) Mitochondrial rates can then be scaled up to the whole-plant level (Atkin & Tjoelker 2003) Thermal acclimation may require changes in the expression of genes that encode respiratory

FIGURE 17 Effects of temperature on plant

respiration (A) Q10of foliar respiration rates

in relation to short-term measurement tem-perature Symbols are the mean Q10of species

of arctic, indicated in blue (49 species), bor-eal, indicated in green (24 species), tem-perate, indicated in brown (50 species), and tropical, indicated in orange (3 species) (B) Assuming a rate of respiration of 0.5 at 08C (arbitrary units), respiration at other tempera-tures was predicted using the linear decline in Q10with increasing temperature (shown in

A) Both the intercept (i.e., R at 08C) and the temperature optimum of respiration (i.e., temperature where respiration rates are max-imal) are shown The lower panels indicate the degree to which respiratory flux is likely lim-ited by enzyme capacity vs substrate supply and adenylates The temperatures where respiratory flux is likely be limited by maxi-mum catalytic enzyme activity (i.e., Vmax) are

indicated in blue (limitations in the cold) and red (limitations at supra-optimal tempera-tures) At moderate temperatures, respiratory flux is likely regulated by the availability of substrate and/or adenylates (i.e., the absolute concentration of ADP and the ratio of ATP:ADP) (after Atkin & Tjoelker 2003; copy-right Elsevier Science, Ltd.)

enzymes or levels of substrates (Sect 4.4) Acclima-tion of leaf respiraAcclima-tion in field-grown Eucalyptus pauciflora (snow gum) occurs without changes in carbohydrate concentrations in leaves (Atkin et al 2000) Temperature acclimation may also be asso-ciated with changes in leaf N concentration, which may affect photosynthesis (Sect 6.1 of Chapter 2A on photosynthesis) and, consequently, the respira-tory energy requirement for phloem loading (Sect 5) (Tjoelker et al 1999) Thermal acclimation in leaves of Arabidopsis thaliana (thale cress) is associated with an increase in rates of O2uptake per unit mitochon-drial protein in mesophyll cells (Armstrong et al 2006)

In addition to the acclimation potential of total respiration, acclimation may also change the parti-tioning of electrons between the cytochrome and alternative pathways as well as the activity of uncoupling proteins Using roots of Triticum aesti-vum(wheat) and Oryza sativa (rice) cultivars with different degrees of respiratory homeostasis, shows that high-homeostasis cultivars maintain shoot and root growth at low temperature (Kurimoto et al 2004a) Irrespective of a cultivar’s capacity to main-tain homeostasis, cytochrome path capacity of intact roots and isolated root mitochondria are lar-ger for plants grown at low temperature, and the maximal activity of cytochrome oxidase show a similar trend In contrast, cyanide-resistant respira-tionof intact roots and relative amounts of alterna-tive oxidase protein in mitochondria isolated from those roots, are lower in high-homeostasis plants grown at low temperature In the roots of low-home-ostasis cultivars, relative amounts of alternative oxi-dase protein are higher at low growth temperature Relative amounts of uncoupling protein show simi-lar trends Maintenance of growth rates in high-homeostasis plants grown at low temperature is obviously associated with both respiratory home-ostasis and a high efficiency of respiratory ATP pro-duction (Kurimoto et al 2004b)

Needles or leaves of cold-hardened plants that maintain relatively low rates of respiration when exposed to higher temperatures maintain higher concentrations of soluble sugars which confers greater frost tolerance During a 58C warmer-than-average winter in north-eastern Sweden, Vaccinium myrtillus(bilberry) may suffer lethal injuries due to the progressive respiratory loss of cryoprotective sugarsfrom their leaves Initial leaf carbohydrate reserves last months only if tissue water content remains high due to frequent misty and rainy days; when dehydrated, the leaves’ cold tolerance increases ( ăOgren 1996) Climate warming may impact significantly on cold hardiness of some

northern European woody plants such as Picea abies(Norway spruce), Pinus sylvestris (Scots pine), and Pinus contorta (lodgepole pine) In lodgepole pine seedlings, needle sugar concentrations may decrease by 15% which makes them more sensitive to frost If the seedlings contain unusually large carbohydrate reserves, as found for Scots pine, these may buffer respiratory expenditure of sugars, and thus avoid frost damage A strong, linear rela-tionship exists between levels of cold hardiness and sugars ( ¨Ogren 2001)

4.6 Low pH and High Aluminum Concentrations

Root respiration rate increases as the pH in the rhizosphere decreases to a level below that at which growth is no longer possible (Fig 18) Net Hỵrelease from roots by Hỵ-ATPase activity is a prerequisite for continued root growth and limits root growth at very low pH values (Schubert et al 1990) One way of coping with excess Hỵuptake at a low pH is to increase active Hỵpumping by plasma-membrane ATPases This increases the demand for respiratory energy (Fig 18) Increased respiration rates can, therefore, allow plants to maintain root growth at noncritical low pH values, by increasing the supply of ATP for Hỵpumping by plasma-mem-brane ATPases

At very low pH values, root growth, net Hỵ release, and respiration rates decline (relative to rates at pH 7.0) The increased entry of Hỵinto the roots under these circumstances appears to be responsible for these effects (Yan et al 1992) Such increased uptake of Hỵtends to disturb cytosolic pH and ultimately root growth The decrease in root respiration at very low pH might, therefore, result from the decreased respiratory demand for growth A low pH may also increase respiration due to the increased solubility of aluminum (Sect 3.1 of Chapter on mineral nutrition) Respiration of intact roots increases in response to aluminum in both aluminum-resistant and sensitive cultivars of Triticum aestivum(wheat) (Collier et al 1993) Root growth and respiration decline at much higher alu-minum concentrations in the resistant than in sensi-tive cultivars (of Sorghum bicolor (sorghum) Tan & Keltjens 1990a,b)

does not occur to any major extent in the sensitive cultivar (Sect 3.1 of Chapter on mineral nutrition)

4.7 Partial Pressures of CO2

CO2concentrations in air pockets in soil are up to 30-fold higher than those in the atmosphere Although respiration rates are highest in superficial layers of soil where root biomass is concentrated, the CO2 concentration increases with increasing profile depth, due to the restricted diffusion of gases in soil pores (Richter & Markewitz 1995)

The CO2concentration in the soil may increase substantially upon flooding of the soil Values of 2.4 and 4.2 mmol CO2mol1(0.24 and 0.42%, respec-tively) occur in flooded soils supporting the growth of desert succulents, as opposed to 0.54 and 1.1 mmol mol1 in the same soils, when well-drained (Nobel & Palta 1989) Good & Patrick (1987) found CO2concentrations of 5.6 and 3.8% in

silt loam, supporting the growth of Fraxinus penn-sylvanica(green ash) and Quercus nigra (water oak), respectively Do such high CO2concentrations affect root respiration?

Root respiration is reversibly inhibited by mmol CO2mol1in two cacti [Opuntia ficus-indica (prickly pear) and Ferocactus acanthodes (compass barrel cactus)] (Nobel & Palta 1989) Full inhibition occurs at 20 mmol CO2mol1(2%) which is irrever-sible if lasting for hours Root respiration of Pseu-dotsuga menziessii (Douglas fir) and Acer saccharum (sugar maple) is also inhibited at soil CO2levels in a range normally found in soil (Qi et al 1994, Burton 1997), whereas no such inhibition occurs for a range of other species (e.g., Bouma et al 1997, Scheurwater et al 1998) Because respiration is only affected by CO2, and not by bicarbonate (Palet et al 1992), the pH of the root environment will greatly affect experimental results (Fig 51 in Chapter 2A on photosynthesis)

How can we account for effects of very high CO2 concentration on respiration? The effects of soil CO2 concentrations on root respiration is probably indir-ect, due to inhibition of energy-requiring processes There may also be direct effects of a high concen-tration of CO2on respiration (i.e., inhibition of cyto-chrome oxidase) (Sect 3.6) Other mitochondrial enzymes are also affected by high concentrations of inorganic carbon (Gonza`lez-Meler et al 1996, Bruhn et al 2007) Malic enzyme, which oxidizes malate to form pyruvate and CO2, is rather strongly inhibited by HCO3 in a range that may well account for inhibition of respiration by CO2as found for some tissues (Chapman & Hatch 1977, Neuberger & Douce 1980) Some of the effects in vitro for several mitochondrial enzymes, however, only appear at CO2 concentrations that are much higher than expected to occur in intact roots

The information in the literature is still too scanty to draw the robust conclusion that CO2levels that normally occur in well drained soil have a direct inhibitory effect on root respiration (Lambers et al 2002) After much discussion on inhibition of leaf respirationby elevated atmospheric CO2 concentra-tions due to global change, there is now wide con-sensus that these are mostly artifacts of the methodology (Jahnke & Krewitt 2002, Davey et al 2004) However, there are indirect effects of long-term exposure of plants to elevated [CO2] These effects are due to changes in, e.g., allocation, plant growth rate, chemical composition of the biomass, rather than accounted for by direct effects (Tjoelker et al 1999, Griffin et al 2001, Davey et al 2004) Across all studies, mass-based leaf dark respiration is reduced by 18%, while area-based leaf respiration

FIGURE18 Effect of the pH in the rhizosphere on (A) net

Hỵrelease, (B) ATP concentration, and (C) respiration

of Zea mays (maize) roots Seedlings were grown at pH 7.0, and either kept at pH 7.0 (open symbols) or exposed to a pH of 4.0 (filled symbols) at the time indicated by the arrow Note that the slopes in A and C give the rate of Hỵ release and respiration (after Yan et al 1992) Copyright American Society of Plant Biologists

is marginally increased (8%), under elevated atmo-spheric CO2concentrations Area-based leaf respira-tion of herbaceous species increases by 28%, but is unaffected in woody species (Fig 19) Mass-based reductions in leaf respiration tend to increase with prolonged exposure to elevated [CO2] In cladodes of Opuntia ficus-indica (prickly pear), reductions in respiration are associated with a decrease in mito-chondrial number and cytochrome path activity, and an increase in activity of the alternative path (Gomez-Casanovas et al 2007) A meta-analysis of published results suggests that the amount of carbon use in leaf dark respiration will increase in a higher-[CO2] environment, because of higher area-based leaf respiration rates and a proportionally greater leaf biomass increase than reductions in mass-based leaf respiration (Wang & Curtis 2002)

4.8 Effects of Plant Pathogens

Pathogen attack on roots or leaves causes an increase in respiration, but the pattern of this respiratory response may differ between sensitive

and resistant varieties of plants For example, nema-tode infection of roots of a susceptible variety of Solanum lycopersicum (tomato) causes root respira-tion first to increase, but then to return to the level of uninfested plants By contrast, the resistant variety shows no initial change in root respiration in response to nematode attack, but after days the respiration rate exceeds that of control plants (Zacheo & Molinari 1987)

Just as with tomato roots, leaves of a susceptible variety of Hordeum distichum (barley) show a large increase in respiration when infected with the fun-guscausing powdery mildew This is expected, as both fungus and host have high demands for energy (the fungus for growth, the host for defense) In the case of barley, most of the respiration is accounted for by host respiration (Farrar & Ryans 1987)

Both mRNA levels that encode the alternative oxidase and the amount of alternative oxidase pro-teinstrongly increase in leaves of Arabidopsis thali-ana (thale cress) that are infiltrated with the leaf-spotting bacterium Pseudomonas syringae (Simons et al 1999) What could be the functional signifi-cance for an increase of this pathway? Pathogenic fungi may produce ethylene and enhance the con-centration of salicylic acid and reactive oxygen spe-cies in the plant (Overmyer et al 2003) These compounds may trigger the increased activity of the alternative path In ripening fruits ethylene enhances alternative respiration; salicylic acid induces the large increase in respiration in the spa-dix of thermogenic Arum species (Sect 3.1) and in vegetative organs of nonthermogenic plants; reac-tive oxygen species trigger expression of the alter-native oxidase in a range of species (Purvis & Shewfelt 1993, Considine et al 2002) Quite likely, the enhanced synthesis of defense-related com-pounds (phytoalexins and other phenolics; Sect of Chapter 9C on effects of microbial pathogens) requires a large production of NADPH in the oxida-tive pentose phosphatepathway (Fig 2) (Shaw & Samborski 1957) This pathway, unlike glycolysis (Fig 3), is not regulated by the demand for meta-bolic energy Products of the oxidative pentose phosphate pathway can enter glycolysis, bypassing the steps controlled by energy demand Additional NADPH can be produced by cytosolic NADP-malic enzyme, which oxidizes malate, producing pyru-vate and CO2 This enzyme is induced upon addi-tion of ‘‘elicitors’’ (i.e., chemical components of a microorganism that induces the synthesis of defense compounds in plant cells) (Sect of Chapter 9C on effects of microbial pathogens) (Schaaf et al 1995) The increased activity of the oxidative pentose path-way and of NADP-malic enzyme probably leads to

FIGURE 19 Long-term effects of elevated atmospheric

CO2on leaf dark respiration expressed on a leaf area

basis (Rda) across 45 independent observations Effects

of growth habit (herbaceous vs woody species) on leaf Rdaresponse and time (length of CO2exposure) on

her-baceous species leaf Rdaresponse to elevated CO2are

the delivery of a large amount of pyruvate and malate to the mitochondria, without there being a large need for ATP As a result, the cytochrome path becomes saturated with electrons, the alternative oxidase is activated (Sect 2.6.2), and much of the electrons are transported via the alternative path-way (Sect 3.3) (Simons & Lambers 1999)

4.9 Leaf Dark Respiration as Affected by Photosynthesis

Both photosynthesis and mitochondrial respiration (‘‘dark’’ respiration, as opposed to photorespiration) produce ATP and NAD(P)H to meet demands for plant growth and maintenance The light reaction in photosynthesis provides ATP and NAD(P)H for bio-synthesis in a leaf cell during illumination, but mito-chondrial respiration in the light is necessary for biosynthetic reactions in the cytosol, such as sucrose synthesis (Kr ăomer 1995) Respiratory activity in the light can be considered part of the photosynthetic process, because it is needed to regulate the redox state of the stroma in the chloroplast during photo-synthesis (Foyer & Noctor 2000) and to maintain the cytosolic ATP pool (Kr ăomer 1995) The rate of mito-chondrial respiration during photosynthesis is therefore determined by the need for this process to provide energy and carbon skeletons in the light Light inhibits leaf ‘‘dark’’ respiration, but the extent of inhibition depends on species and environmental conditions In leaves of Eucalyptus pauciflora (snow gum), respiration is inhibited most at very low light intensities and moderate temperatures, and consid-erably less at higher irradiance The irradiance neces-sary to maximally inhibit R at to 108C is lower than that at 15 to 308C (Atkin et al 2000) In leaves of Xanthium strumarium(common cocklebur) respiration is inhibited at both ambient and elevated CO2 con-centrations, but to a lesser degree for plants grown at elevated (17—24%) than for those grown at ambient (29—35%) CO2 concentrations, presumably because elevated CO2-grown plants have a higher demand fo energy and carbon skeletons (Wang et al 2001) Variations in light inhibition of leaf respiration can have a substantial impact on the proportion of carbon fixed in photosynthesis that is respired

The metabolic origin of the CO2 production in leaf ‘‘dark’’ respiration during photosynthesis can be analyzed by feeding13C-enriched glucose or pyr-uvate to intact leaves Using metabolites that are 13

C-enriched in different positions, reveals that in leaves of Phaseolus vulgaris (common bean) the activ-ity of the TCA cycle is reduced by 95% in the light; pyruvate dehydrogenase activity, however, is much

less reduced (27%) Glucose molecules are scarcely metabolized to liberate CO2 in the light, because glycolysis is down-regulated Instead, glucose is mainly used for sucrose synthesis Several metabolic processes (glycolysis, TCA cycle) are down-regu-lated, leading to a light-dependent inhibition of mitochondrial respiration (Tcherkez et al 2005)

5 The Role of Respiration in Plant Carbon Balance

5.1 Carbon Balance

Approximately half of all the photosynthates pro-duced per day are respired in the same period, the exact fraction depending on species and environ-mental conditions (Table 1) Globally rising tem-peratures tend to increase the proportion of carbon gained in photosynthesis that is subsequently used in respiration (Atkin et al 2007) The level of irradi-ance and the photoperiod appear to affect the car-bon balance of acclimated plants to a relatively small extent, but factors such as inadequate nutrient sup-ply and water stress may greatly increase the pro-portion of photosynthates used in respiration This is accounted for by a much stronger effect of nutri-ents on biomass allocation, when compared to that of irradiance and photoperiod (Chapter on growth and allocation) Root temperature is also likely to affect plant carbon balance because this has a major effect on biomass allocation (Sect 5.2.2 of Chapter on growth and allocation)

5.1.1 Root Respiration

Root respiration accounts for approximately 10—50% of the total carbon assimilated each day in photosynth-esis (Table 1) and is a major proportion of the plant’s carbon budget (Fig 20) This percentage is much higher in slow-growing than in fast-growing plants This is true for a comparison of species that vary in their potential growth rate (Poorter et al 1991) and for plants of the same species that vary in growth rate, due to variation in the nutrient supply (Van der Werf et al 1992a) Root temperatures that enhance biomass allocation to roots (Sect 5.2.2 of Chapter on growth and allocation) probably also increase the proportion of carbon required for root respiration When slow growth is due to exposure to low light levels, however, no greater respiratory burden is incurred (Sect 4.4) To some extent the proportionally greater carbon use in slow-growing plants is accounted for by their

relatively low carbon gain per unit plant mass (Sect of Chapter on growth and allocation) (Poorter et al 1995) This does not explain the entire difference, how-ever; variation in respiratory efficiency and/or respiratory costs for processes like ion transport may play an additional role (Sect 5.2.3)

Root respiration provides the driving force for root growth and maintenance and for ion absorp-tion and transport into the xylem The percentage of total assimilates that are used in root respiration tends to decrease as plants age Such a decrease may be due to a decrease in the demand for respira-tory energy, when the energy required for root growth and ion uptake decreases with increasing age Furthermore, the root mass ratio tends to dec-rease with increasing age, thus decreasing the respiratory burden of roots

The fraction of carbohydrates used in root respiration, including the respiration of symbionts, if present, is affected by both abiotic and biotic environmental factors (Table 1) Root respiration is higher in the presence of an N2-fixing symbiont than when nonnodulated roots are supplied with NO3as a N source This reflects the greater energy requirement for N-assimilation during N2-fixation compared with NO3-assimilation (Sect of Chap-ter 9A on symbiotic associations) The fraction of carbohydrates used in root respiration is also greater

in the presence of a symbiotic mycorrhizal fungus than in nonsymbiotic plants (Table 1)

The proportion of the carbohydrates translocated to roots that is used in respiration, rather than root biomass accumulation, increases with plant age This is primarily due to the increasing role of main-tenance respiration, as root growth slows down and as the quantity of assimilates translocated to roots declines (Sect 5.2) Low nutrient supply also increases the proportion of carbohydrates respired in the roots At a high supply of nutrients, plants respire approximately 40% of the carbon imported into the roots This fraction increases to 60% at very low nutrient supply (Van der Werf et al 1992a) This increase is largely accounted for by a relatively high carbon requirement for maintenance processes com-pared with that in growth processes An additional factor is the proportionally low requirement for root growth (relative to maintenance) under these low-nutrient conditions Finally, specific costs for main-tenance or ion uptake might increase when nutri-ents are in short supply (Van der Werf et al 1994) (Sect 5.2)

5.1.2 Respiration of Other Plant Parts

Leaf respiration provides some of the metabolic energy for leaf growth and maintenance, for ion

FIGURE20 The fraction of all carbohydrates produced

in photosynthesis per day that is consumed in respira-tion as dependent on species and the nitrogen supply Measurements were made on inherently fast-growing (pies on the left) and slow-growing (pies on the right) grass species grown with free nutrient availability (pies at the top) and at a N supply that allowed a relative growth rate of approximately 40 mg g1

day1(pies at the bottom) The percentages at each

transport from the xylem and export of solutes to the phloem Leaf respiration, expressed as a fraction of the carbon gain in photosynthesis, however, varies much less than root respiration, because photo-synthesis, leaf respiration and biomass allocation are affected similarly by changes in nutrient supply This differs from the situation for roots, where a major cause of the large variation found for root respiration (Table 1) is the effect of nutrient supply and genotype on biomass allocation to roots

Rates of photosynthesis and leaf respiration often vary in a similar manner with changes in environ-ment (e.g., N supply and growth irradiance) (Reich et al 1998) This may be explained by greater respiratory costs of export of photosynthates from leaves which vary with the carbon gained in photo-synthesis There may also be greater maintenance costs in leaves with high rates of photosynthesis and high protein concentrations Specific costs for major energy-requiring processes (e.g., for transport of assimilates from the mesophyll to the sieve tubes) may also vary among species and environmental conditions (Cannell & Thornley 2000)

The respiration of other plant parts (e.g., fruits) is largely accounted for by their growth rate and the respiratory costs per unit of growth The mainte-nance component also plays a role In green fruits, a substantial proportion of this energetic requirement may be met by photosynthesis in the fruit (De Jong & Walton 1989, Blanke & Whiley 1995) Respiration of the flowers of Citrus paradisi (grapefruit) shows a distinct peak about 42 days after emergence This peak occurs after a peak in respiration that is asso-ciated with growth of the flower A major part of the respiration of the grapefruit flowers is proba-bly accounted for by the alternative path (Bustan & Goldschmidt 1998, Considine et al 2001)

5.2 Respiration Associated with Growth, Maintenance, and Ion Uptake

The rate of respiration depends on three major energy-requiring processes: maintenance of bio-mass, growth, and (ion) transport, as summarized in the following overall equation:

r ẳ rmỵ cgRGR ỵ ct TR (2)

where r is the rate of respiration (normally expressed as nmol O2or CO2g1s1, but to comply with the units in which RGR is expressed, we use here mmol g1day1); rmis the rate of respiration to produce ATP for the maintenance of biomass; cg (mmol O2or CO2g1) is the respiration to produce

ATP for the synthesis of cell material; RGR is the relative growth rate of the roots (mg g1day1); ct (mol O2 or CO2 mol1) is the rate of respiration required to support TR, the transport rate (mmol g1day1) In roots TR, equals the net ion uptake rate and the rate of xylem loading; in photosynthe-sizing leaves TR equals the rate of export of the products of photosynthesis (from mesophyll to sieve tubes) Although respiration can be measured as either O2uptake or as CO2release, the measure-ments not yield exactly the same values First, RQ may not equal 1.0 (Sect 2.1); second, the rate of CO2 release varies with the rate of NO3 reduction, whereas rates of O2consumption not For this reason O2 consumptionis preferred as a basis to compare plants when we are interested in respira-tory efficiency, whereas CO2 releaseis preferred when comparing the carbon budgets of different plants

By examining these three requirements for res-piratory energy, we can estimate how the ATP pro-duced in respiration is used for major plant functions This equation assumes a tight correlation between the rate of respiration and the rates of major energy-requiring processes; there is no implicit assumption that respiration controls the rate of the energy-requiring processes, or vice versa

5.2.1 Maintenance Respiration

Once biomass is produced, energy must be expended for repair and maintenance Estimates of the costs of maintaining biomass range from 35 to 80% of the photosynthates produced per day (Amthor 2000), higher values pertaining to plants that grow very slowly (Lambers et al 2002) and lower values to shade-adapted species (Noguchi et al 2001b) The energy demands of the individual maintenance processes in vivo are not well known and reliable estimates of individual maintenance costs are scarce A major part of the maintenance energy costs is supposed to be associated with pro-tein turnoverand with the maintenance of solute gradientsacross membranes These costs of main-tenance have been estimated from basic biochemical principles (Penning de Vries 1975, Amthor 2000, Bouma 2005)

In higher plants approximately 2—5% of all the proteins are replaced daily, with extreme estimates being as high as 20% (Van der Werf et al 1992b, Bouma et al 1994) It is quite likely that protein turnover rates vary among plant organs, species and with growth conditions, but the data are too scanty to make firm statements The cost of

synthesizing proteins from amino acids is estimated at 4.7—7.9 ATP, and possibly double that, per peptide bond, or approximately 0.26 (possibly 0.52) g glu-cose g1protein (Amthor 2000) Approximately 75% of amino acids from degraded proteins are recycled (Davies 1979) The remaining 25% must be synthe-sized from basic carbon skeletons, at a cost of 0.43 g glucose g1protein The total cost of protein turn-over is about 28—53 mg glucose g1day1, or 3—5% of dry mass per day Similar calculations for lipids suggest that membrane turnover constitutes a much lower energy requirement, approximately 1.7 mg glucose g1 day1, or 0.2% of dry mass per day Based on an experimentally determined protein half-life of days, the respiratory energy requirement to sustain protein turnover is approximately mmol ATP g1(dry mass) day1[i.e., 7% of the total respira-tory energy produced in roots of Dactylis glomerata (cocksfoot)] Expressed as a fraction of the total main-tenance requirement as derived from a multiple regression analysis (Sect 5.2) [i.e., 2.7 mmol ATP g1(dry mass) day1for Carex (sedge) species], the maintenance requirement for protein turnover is quite substantial (Van der Werf et al 1992b)

Maintenance of solute gradients is also an important maintenance process Some estimates suggest that the cost of maintaining solute gradients are up to 30% of the respiratory costs involved in ion uptake, or approximately 20% of the total respira-tory costs of young roots (Bouma & De Visser 1993) Other processes (e.g., cytoplasmic streaming and turnover of other cellular constituents) are generally assumed to have a relatively small cost Based on these many (largely unproven) assumptions, the total estimated maintenance respiration is approxi-mately 30—60 mg glucose g1day1(3 to 6% of dry mass day1) Measured values of maintenance respiration (8—60 mg glucose g1 day1) suggest that these rough estimates are reasonable

These experimental values for maintenance respiration suggest that protein turnover and the maintenance of solute gradients are by far the lar-gest costs of maintenance in plant tissues If true, then this conclusion has important implications for plant carbon balance because it suggests that any factor that increases protein concentration or turn-over or the leakiness of membranes will increase maintenance respiration

The positive correlation of respiration rate with N concentration (Reich et al 2006) is consistent with the prediction that maintenance respiration depends on protein concentration Thus, leaves that have a high N investment in Rubisco and other photosynthetic enzymes have a correspondingly high maintenance respiration Whether this is a general phenomena

remains to be investigated (Van der Werf et al 1992b) Higher respiration rates might also reflect greater costs for the loading of photosynthates in the phloem, which is an ATP-requiring process (Sect 3.3 of Chapter 2C on long-distance transport) Whatever the explanation for the higher leaf respiration rates, they contribute to their higher light-compensation point (Sect 3.2.1 of Chapter 2A on photosynthesis) and, therefore, place a higher limit on the irradiance level at which these leaves can maintain a positive carbon balance Thus, there is a trade-off between high meta-bolic activity (requiring high protein concentrations and rapid loading of the phloem) and the associated increase in cost of maintenance and transport

The stimulation of maintenance respiration by temperature is a logical consequence of the increased leakage and of protein turnover that occurs at high temperature (Rachmilevitch et al 2006) This pro-vides a conceptual framework for studies that seek to explain why different tissues and species differ in their Q10of respiration Perhaps this reflects differ-ences in membrane properties upon prolonged exposure to higher temperatures or in thermal stabi-lity of proteins, with corresponding differences in protein turnover (Criddle et al 1994) It might also reflect a difference in contribution of the cytochrome and the alternative pathways

Increased maintenance respiration is often ass-umed to be the cause of declines in forest productivity in late succession (e.g., Waring & Schlesinger 1985) Maintenance respiration remains relatively constant through succession, however, while growth respi-ration declines (Fig 21) The more likely cause of

FIGURE21 Construction costs of leaf biomass Most of

reduced growth in old forest stands is a reduced carbon gain caused by loss of leaf area and loss of photosynthetic capacity associated with reduced hydraulic conductance and in some cases with reduced nutrient availability (Ryan et al 1997)

5.2.2 Growth Respiration

Production of biomass (biosynthesis) requires the input of carbohydrates, partly to generate ATP and NAD(P)H for biosynthetic reactions and partly to provide the carbon skeletons present in biomass (Fig 22; Table 9) Plant tissue is, in general, more reduced than the carbohydrates from which it is produced, and the cost of biosynthesis from pri-mary substrates must therefore include the carbo-hydrates necessary to supply reducing power, for example for the reduction of NO3 If a more reduced source of N is absorbed instead (e.g., NH4ỵ or amino acids) (Sect 2.2 of Chapter on mineral nutrition), then biosynthetic costs are less When a tissue senesces, most of the chemical consti-tuents are lost to the plant, but some are resorbed and can be used in the production of new tissues The final cost of producing a tissue is the initial cost minus resorption (Fig 23)

In photosynthetically active leaves, some of the metabolic energy (ATP and NAD(P)H) may come directly from photosynthesis In heterotrophic tis-sues such as roots, and in leaves in the dark, respiration provides the required energy The amount of respiratory energy that is required for biosynthesis can be calculated from the composi-tion of the biomass in several ways, as discussed in this section

First, costs for biosynthesis can be derived from detailed information on the biochemical

composition, combined with biochemical data on the costs of synthesis of all the major compounds: protein, total nonstructural carbohydrates (i.e., sucrose, starch, fructans), total structural carbohy-drates (i.e., cellulose, hemicellulose), lignin, lipid, organic acids, minerals This can be extended to include various other compounds, e.g., soluble amino acids, nucleic acids, tannins, lipophilic defense compounds, alkaloids, but these are mostly ignored and generally combined with the major ones Taking glucose as the standard substrate for biosynthesis, one can estimate the amount of glu-cose required to provide the carbon skeletons, redu-cing equivalents and ATP for the biosynthesis of plant compounds in tissues (Table 9; Poorter & Vil-lar 1997)

Note that the amount of product produced per unit carbon substrate (production value, PV’) varies nearly threefold among chemical constituents (Table 10), with lipid and lignin being ‘‘most expen-sive’’ (i.e., requiring greatest glucose investment per gram of product), and organic acids ‘‘least expen-sive’’ Compounds like proteins and lipids are very costly in terms of ATP required for their biosynth-esis, whereas carbohydrates and cellulose are not There are both expensive and cheap ways to pro-duce structure in plants (lignin and cellulose/hemi-cellulose, respectively) and to store energy (lipid and sugars/starch, respectively) (Chapin 1989) Plants generally use energetically cheap structural components (cellulose/hemicellulose) and energy stores (sugars and starch) By contrast, mobile ani-mals and small seeds, where mass is an important issue, often use lipids as their energy store Immo-bile animals, like plants, use carbohydrate (glyco-gen) as their primary energy store Knowing the costs and concentrations of the major compounds

FIGURE22 Fate of carbon that is initially invested (CI) in

synthesizing a structure Some of the carbon is retained in the biomass (CB), the remainder is required for

respiration (CR) Of the carbon in the biomass (CB),

most is lost or respired when a plant part is shed (CS)

but some is resorbed (CP) for subsequent use (after

Chapin 1989)

TABLE9 Values for characterizing the conversion of substrates to products during biosynthesis, excluding costs of substrate uptake from the environment.*

Compound PV’ ORF’ CPF’ RQ’ HRF ERF

Amino acids with NH4ỵ 700 169 5772 34 11.2 –1.4

Amino acids with NO3 700 169 5772 34 26.7 39.0

Protein with NH4ỵ 604 163 5727 35 –12.9 34.9

Protein with NO3 604 163 5727 35 31.4 82.0

Carbohydrates 853 1295 – –3.6 12.2

Lipids 351 10705 – –10.1 51.0

Lignin 483 1388 5545 –4.3 18.7

Organic acids 1104 –1136 – 16.9 –4.5

Source: De Visser et al (1992)

*Production Value, PV’: mg of the end product per g of substrate required for carbon skeletons and energy production, without

taking into account the fate of excess or shortage of NAD(P)H and ATP (the term Production Value, PV, is used when PV’ is corrected for this component); Oxygen Requirement Factor, ORF’: mmol of O2consumed per gram of substrate required for carbon

skeletons and energy production, without taking into account the fate of excess or shortage of NAD(P)H and ATP; Carbon dioxide Production Factor, CPF’: mmol of CO2produced per g of substrate required for carbon skeletons and energy production, without

taking into account the fate of excess or shortage of NAD(P)H and ATP (the term Carbon dioxide Production Factor, CPF, is used when CPF’ is corrected for this component); RQ’ is the ratio of CPF’ and ORF’; Hydrogen Requirement Factor, HRF: moles of NAD(P)H required (–) or produced (+) per gram of end product; Energy Requirement Factor, ERF: moles of ATP required (–) or produced (+) per gram of end product (Penning de Vries et al 1974) More recent findings, for example on the importance of targeting sequences of proteins which are required to ‘‘direct’’ the synthesized proteins to a specific compartment in the cell, indicate that the costs for protein synthesis are likely to be substantially, possibly even double the value as presented in this table

FIGURE23 The chemical composition and carbon cost of

producing leaves and stems of 13 species of tundra plants Species shown are Salix pulchra (willow, Sp), Betula nana (dwarf birch, Bn), Vaccinium uliginosum (blueberry, Vu), Arctostaphylos alpina (bearberry, Aa), Rubus chamaemorus (cloudberry, Rc), Pedicularis capi-tata (wooly lousewort, Pc), Ledum decumbens

in plant biomass, we can calculate the costs for a gram of biomass As for individual compounds, these costs can be expressed in terms of glucose, O2requirement, CO2release, requirement for redu-cing power and ATP (Table 10)

The major assumption underlying the approach based on the biochemical composition of the biomass is that glucose is the sole substrate for all ATP, reduc-tant, and carbon skeletons When some of these resources are derived directly from photosynthesis, costs may be lower Costs may be higher when the alternative path, rather than the cytochrome path plays a predominant role in respiration If we restrict this approach to nonphotosynthetic tissues in which the contribution of the cytochrome and alternative respiratory pathway is known, then there is still a source of error, if these tissues import compounds other than glucose, for example amino acids, as a substrate for biosynthesis

A second method for estimating the construction cost is based on information on the elemental com-positionof tissues: C, H, O, N, and S (McDermitt & Loomis 1981) The constructions costs that are not covered by this equation include costs of mineral uptake and transport of various compounds in the plant, costs for providing ATP for biosynthetic reac-tions, and reductant required to reduce molecular oxygen in some biosynthetic reactions This method is less laborious than the first method, which requires detailed chemical analysis; however, it is based on the observations of the first method (i.e., that expensive compounds are generally more reduced than glucose, whereas cheap compounds are more oxidized) (Poorter 1994) Although this method, based on elemental analysis of plant bio-mass, may seem a crude approach, the approach is

surprisingly effective First, this is because two thirds of the construction costs are costs to provide carbon skeletons rather than for respiration Second, most of the carbon that does not end up in the carbon skeletons of biomass is required to reduce carbon skeletons, and not for the production of ATP So, even in the absence of detailed information on respiratory pathways, construction costs can be esti-mated rather accurately In fact, the second method can be simplified even further, taking into account only the carbon and ash content of biomass and ignoring minor constituents that have only a small effect on the production value (Vertregt & Penning de Vries 1987)

The level of reduction of plant biomass is approximately linearly related to its heat of com-bustionas well as its costs of construction (McDer-mitt & Loomis 1981) For example, lipids are highly reduced compounds and have a high heat of com-bustion A third method, therefore, uses this approx-imation to arrive at costs for providing carbon skeletons and reductant for biosynthesis (Williams et al 1987)

Given the three-fold range in the cost of produ-cing different organic constituents in plants and the large range in concentrations of these constituents among plant parts and species [often 2—10-fold (Fig 24)], we might expect large differences in costs of synthesizing tissues of differing chemical composition A given tissue, however, tends to have either a high concentration of proteins and tannins (allowing high metabolic activity and che-mical defense of these tissues) or a high concentra-tion of lignin and lipophilic secondary metabolites (Chapin 1989) The negative correlation between the concentrations of these two groups of TABLE 10 An example of a simplified calculation of the variables characterizing biosynthesis of biomass from glucose, nitrate and minerals

Compound

Concentration in biomass required(mg g1

dry mass)

Glucose for synthesis

O2

required for synthesis

(mmol)

CO2production

during synthesis (mmol)

NAD(P)H required for

synthesis (mmol)

ATP required for synthesis (mmol)

N-compounds 230 371 65 2100 7.14 17.83

Carbohydrates 565 662 857 2.03 6.92

Lipids 25 71 807 0.25 1.27

Lignin 80 166 230 918 0.34 1.50

Organic acids 50 45 52 0.84 0.23

Minerals 50 0 0

Total 1000 1315 295 4630 3.68 27.29

Source: Penning de Vries et al (1974)

expensive constituents is seen in the comparison of leaves vs stems or in the comparison of leaves of rapidly growing species (e.g., forbs) and slowly growing species (evergreen shrubs) (Fig 24) The net result of this trade-off between expensive com-ponents allowing rapid metabolic activity (pro-teins) vs those allowing persistence (lignin and lipophilic defensive compounds) is that the cost of all plant species and plant parts are remarkably similar: approximately 1.5 g glucose per gram of biomass (Figs 23 and 24) Another important cor-relation that explains the similarity of construction costs across species and tissues is that tissues of fast-growing species that have high protein con-centrations (an expensive constituent) also have high concentrations of minerals (cheap constitu-ents) (Poorter 1994, Villar et al 2006) This explains why extremely simple relationships are excellent predictors of costs of synthesis The similarity of

cost of synthesis across species, plant parts and environments (Chapin 1989, Villar et al 2006) dif-fers from early conclusions that emphasized the high costs associated with lipids and lignin in ever-green leaves (Miller & Stoner 1979)

Small seeds are an exception to the generaliza-tion that all plant biomass has a similar cost of synthesis, because seed lipids are primarily an energy store (rather than an antiherbivore defense) and are positively associated with protein concen-tration (Fig 25), leading to a high carbon cost The similarity among species and tissues in carbon cost of synthesis has the practical consequence that biomass is an excellent predictor of carbon cost One possible ecological explanation for this pat-tern is that carbon is such a valuable resource that natural selection has led to the same minimal car-bon cost for the construction of most plant parts An alternative, and more probable, explanation is that the negative correlations among ex-pensive constituents and the positive correlation between protein and minerals have a basic physiological significance that, by coincidence, leads to a similar carbon cost of synthesis in most structures For example, lignin and protein concentrations may be negatively correlated because young expanding cells have a high protein concentration, but cell expansion would be prevented by lignin, or that heavy lignification might render cell walls less permeable to water and solutes which would be disadvantageous in tissues with high metabolic activity (as gauged by high protein concentration) In general, currently available data suggest that costs of synthesis differ much less within (10—20%) and among (25%) ecosystems than other causes of variation in carbon balance, such as respiration and allocation (Chapin 1989, Villar et al 2006)

FIGURE24 Range of construction costs for a survey of

leaves (n = 123), stems (n = 38), roots (n = 35), and fruits/ seeds (n = 31) Values are means and 10th and 90th percentiles (Poorter 1994) Copyright SPB Academic Publishing

FIGURE 25 Annual carbon use for stem and

5.2.3 Respiration Associated with Ion Transport

Ion transport across membranes may occur via ion channels, if transport is down an electroche-mical potential gradient, or via ion carriers, which allow transport against an electrochemical potential gradient (Sect 2.2.2 of Chapter on mineral nutrition) Because cation transport from the rhizosphere into the symplast mostly occurs down an electrochemical potential gradi-ent, cation channels are often involved in this transport This requires respiratory energy to extrude protons into the apoplast and create an electrochemical potential gradient Transport of anions from the rhizosphere into the symplast almost invariably occurs against an electrochemical gradient and hence requires respiratory energy, mostly because such anion transport is coupled to proton re-entry into the cells (Sect 2.2.2 of Chapter on mineral nutrition)

The situation is exactly the opposite for the trans-port of ions from the symplast to the xylem (xylem loading) Anions might enter the xylem via chan-nels, as this transport is mostly down an electroche-mical gradient; however, we know little about such a mechanism (De Boer & Wegner 1997) The trans-port of most cations is against an electrochemical gradient, and hence the transport of cations to the xylem depends directly on metabolic energy Release of anions into the xylem may be passive, but it still depends on the presence of an electroche-mical potential gradient, which is maintained by the expenditure of metabolic energy On the other hand, resorption of anions must be active (involving car-riers) whereas that of cations may occur via chan-nels (Wegner & Raschke 1994, De Boer & Wegner 1997)

When NO3is the major source of N, this will be the major anion absorbed, because only 10% and 1%, respectively, as much P and S compared with N are required to produce biomass (Fig 33 in Sect 4.1.1 of Chapter on mineral nutrition) Uptake of amino acids will also be against an electrochemical potential gradient and hence require a proton-cotransport mechanism similar to that described for NO3 Like the uptake of NO3and amino acids, P uptake also occurs via a proton symport mechanism (Sect 2.2.2 of Chap-ter on mineral nutrition) When P availability is low, however, P acquisition may require exuda-tion of carboxylates (Sect 2.2.5 of Chapter on mineral nutrition) which will incur additional car-bon expenditure Similarly, P acquisition through a symbiotic association with mycorrhizal fungi

requires additional carbon (Sect 2.6 of Chapter 9A on symbiotic associations)

As long as there is an electrochemical potential gradient, which is a prerequisite for the uptake of anions, cations can enter the symplast passively In fact, plants may well need mechanisms to excrete cations that have entered the symplast passively, to avoid excessive uptake of some cation (e.g., Naỵ) (Sect 3.4.2 of Chapter on mineral nutrition) When NH4ỵ is the predomi-nant N source for the plant, such as in acid soils where rates of nitrification are low, this can enter the symplast via a cation channel Rapid uptake of NH4ỵ, however, must be balanced by excretion of Hỵ, so as to maintain a negative membrane poten-tial Hence, NH4ỵuptake also occurs with expen-diture of respiratory energy

When NO3is the predominant N-source, rather than NH4ỵor amino acids, there are additional costs for its reduction These show up with carbon costs and CO2 release, but not in O2 uptake (Table 9), because some of the NADH generated in respiration is used for the reduction of NO3rather than for the reduction of O2 As a result, the RQ strongly depends on the source of N (NH4ỵor NO3; Table 2) and on the rate of NO3reduction Costs asso-ciated with NO3 acquisition are less when the reduction of NO3occurs in leaves exposed to rela-tively high light intensities, as opposed to reduction in the roots, because the reducing power generated in the light reactions exceeds that needed for the reduction of CO2 in the Calvin cycle under these conditions (Sect 3.2.1 of Chapter 2A on photosynthesis)

Given that N is a major component of plant bio-mass, most of the respiratory energy associated with nutrient acquisition in plants with free access to nutrients will be required for the uptake of this nutrient

5.2.4 Experimental Evidence

Measurements made with roots provide an oppor-tunity to test the concepts of maintenance respira-tion, growth respirarespira-tion, and respiration associated with transport We assume that the rate of respira-tion for maintenance of root biomass is linearly related to the root biomass to be maintained Sec-ond, we assume that the rate of respiration for ion transport is proportional to the amount of ions taken up, whereas that for root growth is propor-tional to the relative growth rate of the roots, provided the chemical composition of the root bio-mass does not change in a manner that affects the

specific costs of biomass synthesis; superimposed is the maintenance respiration Third, we assume that the contribution of the alternative path to total respiration is constant Based on these assumptions, which are largely untested, the rate of ATP production per gram of roots and per day can be related to the relative growth rate of the roots and the rate of anion uptake by the roots We can improve the approach by assessing the contri-bution of the alternative path (Box 2B.1), and cor-rect for any changes during plant development (Florez-Sarasa et al 2007) The costs of the three processes can then be estimated by multiple regression analysis, presented graphically in a three—dimensional plot (Fig 26, left) If a plant’s relative growth rate and rate of anion uptake are very closely correlated, which is common, then a multiple regression analysis cannot separate the costs of growth from those of ion uptake (Fig 26, right)

Using the analysis as depicted in Fig 26A, respiratory costs for growth, maintenance, and ion uptake have been obtained for a limited number of species (Table 11A) Quite often, the correlation between relative growth rate and nutrient uptake is so tight, that a linear regression analysis, as depicted in Fig 26B, is the only approach pos-sible (Table 11B) There is quite a large variation

in experimental values among species This may reflect real differences between species; however, the variation may also indicate that the statistical analysis ‘‘explained’’ part of respiration by ion uptake in one experiment and by maintenance in another For example, a costly process like ion leakage from roots, followed by re-uptake, may show up in the slope or in the y-intercept in the graph, and suggest large costs for ion uptake or for main-tenance, respectively At the highest rates of growth and ion uptake (young plants, fast-grow-ing species) these data suggest that respiration for growth and ion uptake together account for about 60% of root respiration, and that mainte-nance respiration is relatively small With increasing age, when growth and ion uptake slow down, maintenance respiration accounts for an increasing proportion of total respiration (over 85%)

The specific costs for Carex (sedge) species (Table 11A) were used to calculate the rate of root respiration of 24 other herbaceous species of differing potential growth rate whose rates of growth and ion uptake were known These calcula-tions greatly over-estimate the rate of root respira-tion of fast-growing species, when compared with measured values (Fig 27) This suggests that

FIGURE26 (A) Rate of O2consumption per unit fresh

mass (FM) in roots as related to both the relative growth rate (RGR) of the roots and their net rate of anion uptake (NIR) (B) Rate of O2consumption per

unit fresh mass in roots as related to the relative growth rate of the roots The plane in (A) and line in (B) give the predicted mean rate of O2consumption The intercept

of the plane in (A) and the line in (B) with the y-axis

gives the rate of O2consumption in the roots which is

required for maintenance The slope of the projection of the line on the y–z plane gives the O2consumption

either the efficiency of respiration is greater (e.g., relatively more cytochrome path and less alter-native path activity) in fast-growing species, or that the specific costs for growth, maintenance or ion

uptake are lower for fast-growing species Is there any evidence to support either hypothesis?

Roots of fast-growing grass species exhibit higher rates of alternative path activity than slow-growing grasses (Millenaar et al 2001), and hence there is no evidence for a more efficient respiration in roots of fast-growing species Specific respi-ratory costs for root growth are somewhat higher for fast-growing species (Fig 28A), and maintenance costs, if anything, are higher, rather than lower, for roots of fast-growing species, possibly be due to their higher protein concentra-tions and associated turnover costs (Scheurwater et al 1998, 2000) If neither a low respiratory efficiency nor higher costs for growth or mainte-nance can account for unexpectedly fast respira-tion rates of slow-growing plants, then the

discrepancy between the expected and

measured rates of root respiration (Fig 27) must be based on higher specific costs for ion uptake in the inherently slow-growing species (Fig 28B) These higher specific costs when plants are grown with free access to NO3are accounted for by a large efflux of NO3-(Sect 2.2.2 of Chapter on mineral nutrition; Scheurwater et al 1999) It should be noted that many slow-growing species naturally grow in a low-NO3 TABLE11 (A) Specific respiratory energy costs for the maintenance of root biomass, for root growth and for ion uptake (B) Specific respiratory energy costs for the maintenance of root biomass and for root growth including costs for ion uptake

Carex species Solanum tuberosum Zea mays

A

Growth, 6.3 10.9 9.9

mmol O2(g dry mass)1

Maintenance, 5.7 4.0 12.5

nmol O2(g dry mass)1s1

Anion uptake, 1.0 1.2 0.53

mol O2(mol ions)1

B

Dactylis glomerata Festuca ovina Quercus suber Triticum aestivum

Growth + ion uptake, 11 19 12 18

mmol O2(g dry mass)1

Maintenance, 26 21 22

nmol (g dry mass)1s1

Sources: (A) The values were obtained using a multiple regression analysis, as explained in Figure 25A [Van der Werf et al 1988: average values for Carex acutiformis (pond sedge) and Carex diandra (lesser panicled sedge); Bouma et al 1996: Solanum tuberosum (white potato); Veen 1980: Zea mays (corn)] (B) The values were obtained using a linear regression analysis, as explained in Figure 25B [Scheurwater et al 1998: Dactylis glomerata (cocksfoot) and Festuca ovina (sheep’s fescue); Mata et al 1996: Quercus suber (cork oak); Van den Boogaard, as cited in Lambers et al 2002: Triticum aestivum (wheat)]

FIGURE27 The rate of root respiration of fast-growing

and slow-growing herbaceous C3 species The broken

line gives the calculated respiration rate, assuming that specific costs for growth, maintenance, and ion uptake are the same as those given in Table 11 and identical for all investigated species (Poorter et al 1991) Copyright Physiologia Plantarum

environment, and hence would rarely be exposed to the experimental conditions as used referred to here The question that remains to be addressed is whether NO3efflux also plays a role when NO3 availability limits plant growth Given that root respiration rates are also unexpectedly high for plants grown at a severely limiting NO3supply (Fig 14), this is certainly a likely possibility

The rate of root respiration of plants grown with a limiting nutrient supply is lower than that of plants grown with free access to nutrients, but not nearly as low as expected from their low rates of growth and nutrient acquisition (Sect 4.3) This again suggests increased specific costs, possibly for ion uptake Further experimental evidence is needed to address this important question concern-ing the carbon balance and growth of slow-growconcern-ing plants

In summary, experimental data suggest that the concept of respiration associated with growth, maintenance, and ion uptake is a valuable tool in understanding the carbon balance of plants and that the partitioning of respiration among these func-tions may differ substantially with environment and the type of plant species

6 Plant Respiration: Why Should It Concern Us from an Ecological Point of View?

A large number of measurements have been made on the gas exchange (i.e., rates of photosynthesis, respiration, and transpiration) of different plants growing under contrasting conditions Those mea-surements have yielded fascinating experimental results, some of which have been discussed in Chapter 2A on photosynthesis There is often the (implicit) assumption, however, that rates of photo-synthesis provide us with vital information on plant growth and productivity Certainly, photosynthesis is essential for most of the gain in plant biomass; however, can we really derive essential information on growth rate and yield from measurements on photosynthesis alone?

Rates of photosynthesis per unit leaf area are poorly correlated with rates of growth, let alone final yield (Evans 1980) One of the reasons that have emerged in this chapter on plant respiration is that the fraction of all carbohydrates that are gained in photosynthesis and subsequently used in respiration varies considerably First, slow-growing genotypes require relatively more of their photoassimilates for respiration Secondly, many environmental variables affect respiration more than photosynthesis This is true both because respiration rate is sensitive to environment and because the size of nonphotosynthetic plant parts, relative to that of the photosynthetically active leaves depends on the environment, as dis-cussed in Chapter on growth and allocation Clearly, an important message from this chapter on plant respiration should be that measurements of leaf photosynthesis by themselves cannot pro-vide us with sound information on a plant’s growth rate or productivity

A second message worth emphasizing here is that respiration and the use of respiratory energy [NAD(P)H, ATP] are not as tightly linked as long believed Respiration may proceed via pathways that not yield the respiratory products needed

FIGURE28 Characteristics of root respiration of

for growth, but produces heat instead These com-ponents of respiration can be substantial, at least in some plants under some conditions In specific tis-sues the production of heat may be of use to the plant, but the ecophysiological significance of it in other tissues is different

A challenge for the future will be to explore to what extent respiration scales with other plants traits, as has been done for photosynthesis (Sect of Chapter 2A) There is clear evidence that specific respiration rates scale with tissue N con-centration, just like photosynthesis does, but we have yet to explore scaling patterns with a range of other traits

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2

Photosynthesis, Respiration, and Long-Distance Transport

2C. Long-Distance Transport of Assimilates

1 Introduction

The evolution of cell walls allowed plants to solve the problem of osmoregulation in freshwater envir-onments; however, cell walls restrict motility and place constraints on the evolution of long-distance transport systems Tissues are too rigid for a heart-pump mechanism; instead, higher plants have two systems for long-distance transport The dead ele-ments of the xylem allow transport of water and solutes between sites of different water potentials That transport system is dealt with in Chapter on plant water relations The other transport system, the phloem, allows the mass flow of carbohydrates and other solutes from a source region, where the hydrostatic pressure in the phloem is relatively high, to a sink region with lower pressure

Plants differ markedly in the manner in which the products of photosynthesis pass from the meso-phyll cells to the sieve tubes (phloem loading) through which they are then transported to a site where they are unloaded and metabolized (Fig 1) Plants also differ with respect to the major carbon-containing compounds that occur in the sieve tubes, which is the complex consisting of sieve elements and companion cells For reasons that are explained in this chapter, there is a close association between the type of phloem loading (symplastic or apoplas-tic) and the type of major carbon compound (sucrose or oligosaccharides) transported in the

phloem Sucrose is a sugar composed of two hexose units, whereas an oligosaccharide comprises more than two units In addition, there appears to be an association between the pattern of phloem loading (symplastic vs apoplastic) and the ecological distri-bution of species and between phloem structure and plant habit (vine vs tree or shrub) It is this associa-tion between phloem transport and ecological adap-tation that we explore in this chapter

2 Major Transport Compounds in the Phloem: Why Not Glucose?

In animals, glucose is the predominant transport sugar, albeit at much lower concentrations than those of predominant sugars in the sieve tubes of higher plants In plants, sucrose is a major constitu-ent of phloem sap, whereas glucose and other monosaccharides are found only in trace concentra-tions Why not glucose?

such as glucose and fructose, which contain an alde-hyde group, which is readily oxidized to a car-boxylic acid group; hence they are known as reducing sugars A good transport compound should also be protected from enzymatic attack until it arrives at its destination In this way the flow of carbon in plants can be controlled by the presence of key hydrolyzing enzymes in appropri-ate sink tissues Thus, sucrose appears to be a preferred compound because it is ‘‘protected’’

Other ‘‘protected’’ sugars include the oligosac-charides of the raffinose family: raffinose, stachyose, verbascose These sugars are formed by the addition of one, two or three galactose molecules to a sucrose molecule (Fig 2) They are major transport sugars in a wide range of species Other transport compounds are the sugar alcohols (sorbitol, mannitol, dulcitol) (Fig 2), e.g., in Apiaceae [e.g., Apium graveolens (celery)], Rosaceae [e.g., Prunus persica (peach)], Combretaceae, Celastraceae, and Plantaginaceae, and oligofructans [e.g., in Agave deserti (century plant)] (Wang & Nobel 1998) Despite the diversity in composition of the phloem transport fluid among species, nearly all species are similar in their very

low concentrations of monosaccharides (glucose, fructose) (Turgeon 1995)

In addition to sugars, phloem sap contains a range of organic acids, amino acids, and inorganic ions Concentrations of Ca, Fe, and Mn in the phloem sap are invariably low; this may be related to the fact that these nutrients tend to precipitate at the relatively high pH that characterizes phloem sap (Fig 6.1B in Chapter on mineral nutrition) As a result, growing leaves and fruits must predomi-nantly import these nutrients via the xylem If the Ca concentrations in the xylem sap and the tran-spiration rates are low, some fruits [e.g., of Solanum lycopersicum(tomato) and Capsicum annuum

(capsi-cum)] may show Ca-deficiency symptoms

(Marschner 1995) Similarly, legume seeds may show seed disorders when the import of Mn becomes too low, and calcifuge species show yel-lowing of their youngest leaves, due to a restricted uptake of Fe at high soil pH (Sect 2.2.6 of Chapter on mineral nutrition) That is, plant organs that predominantly import specific nutrients via the xylem may show deficiency symptoms when tran-spiration rates are low, when the concentration of

FIGURE1 Sucrose and other products of photosynthesis (photosynthates) are generated in palisade (PMC) and spongy (SMC) mesophyll cells They are either symplas-tically (top) or apoplassymplas-tically (bottom) moved to the companion cells (CC) and/or sieve elements (SE) of the minor vein phloem and are subsequently exported to sink regions of the plant Plasmodesmata connect all cell types, but the roles they play in the various transport steps differ in different species In particular, the num-ber of plasmodesmata connecting bundle sheath cells (BSC) to companion cells varies greatly In some plants there are many, as depicted at the top In others there

are relatively few, as shown at the bottom The ultra-structure and biochemistry of the companion cells in minor veins also differs considerably in different plants, an indication of different loading strategies (as discussed in the text) The plasmodesmata between companion cells and sieve elements are especially wide and accom-modate the passage of much larger molecules Once inside the sieve elements, photosynthates are carried away in the export stream The minor veins merge to create larger veins with connected sieve tubes (ST) Though not depicted here, all sieve elements have adjoining companion cells

these specific nutrients in the xylem is very low, due to restricted uptake, or both

Most plant viruses can also move over long dis-tances in the phloem (e.g., Roberts et al 1997) More-over, alarm signals involved in induced systemic resistance, hormones, and microRNA (miRNA) molecules, which are a class of developmental sig-naling molecules, are also transported via the phloem (Van Bel 2003, Juarez et al 2004, Lough & Lucas 2006)

3 Phloem Structure and Function

In the process of transporting assimilates from the site of their synthesis (the source) to the site where they are used (the sink), the products of photo-synthesis must move from the mesophyll cells to the transport system: the sieve elements Sieve elements are living cells with characteristic sieve

areas in their cell walls When pores connect adja-cent cells, they are commonly differentiated into sieve plates, with pores ranging in diameter from 1—15 mm

In the gymnosperm Sequoiadendron giganteum (giant redwood) the source-sink distance of the phloem path can be as much as 110 m, due to the enormous height of the tree This example is extreme, because sinks mostly receive assimilates from adjacent source leaves, but it illustrates the point that transport sometimes occurs over vast dis-tances, for example to growing root tips far removed from source leaves Long-distance transport in the phloem occurs by mass flow, driven by a difference in hydrostatic pressure, created by phloem loading in source leaves and unloading processes in sink tissues

When sieve tubes are damaged and the pressure declines, sieve plates tend to be blocked Short-term sealing mechanisms are triggered by Ca and involve

FIGURE The chemical structure of the major sugars

and some sugar alcohols transported in sieve tubes

proteins, e.g., forisomes in legumes (Furch et al 2007) Long-term sealing involves blocking with a glucose polymer, callose

3.1 Symplastic and Apoplastic Transport

How are the products of photosynthesis in the meso-phyll loaded into the sieve tubes? There are two ways in which solutes can pass from one plant cell to another One is through plasmodesmata This is known as symplastic (or symplasmic) transport (The symplastis the internal space of cells, surrounded by plasma membranes Since plasmodesmata are lined by the plasma membrane, cells connected by plasmodesmata form a symplastic continuum.) Solute passage through plasmodesmatal channels is passive, unassisted by proteins that mediate active transport Therefore, symplastic transport cannot, by itself, establish a solute concentration gradient

The second route available for solute movement from one cell to another is through the apoplast (The apoplast is the space outside the plasma mem-branes, including the cell walls and the xylem con-duits.) If solute molecules originate inside cells, as photosynthates do, then apoplastic (or apoplasmic) transport involves release of the solute from the symplast into the cell wall space, followed by uptake into recipient cells The uptake step may involve specific transporters located in the plasma membrane, and often occurs against a concentration

gradient In some cells that are responsible for high solute flux the walls are invaginated to increase the surface area of the plasma membrane and uptake capacity These are known as transfer cells (Offler et al 2003) Uptake may also occur nonselectively by endocytosis (Samaj et al 2004)

Since the solute concentration of the phloem is often much higher than that of the mesophyll tissue, it is not surprising that, in many plants, sucrose is loaded into the phloem from the apoplast However, in other plants, photoassimilate molecules follow an entirely symplastic pathway into the phloem (Sect 3.3)

3.2 Minor Vein Anatomy

The veins in leaves of dicotyledonous species branch progressively to form a reticulate network Up to six or seven branching classes (orders) can be recognized in some species The largest vein is the midrib (class I) and the smallest few classes are called minor veins Minor veins are much more extensive than the major veins, and thoroughly permeate the mesophyll tissue Few mesophyll cells are more than or cells away from a minor vein Clearly, the minor venation is responsible for most, if not all, phloem loading of photoassimilates Since structure is often a meaningful guide to function, the comparative anatomy of the minor veins should provide clues to the mechanisms of

FIGURE (Left) Minor vein from sink-source transition

region of a leaf of Cucumis melo (melon) Abaxial phloem contains two intermediary cells (I) and imma-ture sieve elements (not labeled) adjacent to a parench-yma cell (P) The interface of intermediary cells and bundle-sheath cells is indicated by arrows A developing tracheid (T) and adaxial companion cell (CC) with its

immature sieve element (not labeled) are also present Bar = mm (Volk et al 1996) (Right) Transverse section of a typical Arabidopsis thaliana (thale cress) minor vein, with five sieve elements BS, bundle sheath cell; CC, companion cell; PP, phloem parenchyma cell; SE, sieve element; T, tracheary element; VP, vascular par-enchyma cell Bar = mm (Haritatos et al 2000)

the loading process in different species Gamalei (1989, 1991) studied the minor-vein anatomy of over 1000 higher plant species He recognized dif-ferent degrees of plasmodesmatal connectivity between the mesophyll cells and the minor vein phloem in different species In the herb Senecio ver-nalis (eastern groundsel) the frequency is around 0.03 plasmodesmata mm—2interface area, against 60 in the tree Fraxinus ornus (manna ash) Gamalei grouped plants into arbitrarily defined types Type plants exhibit about three orders of magnitude more plasmodesmatal contacts than type 2, while intermediates between the two extremes (types 1-2a) differ by about one to two orders of magnitude in plasmodesmatal frequency Within Gamalei’s types there are subgroups Type plants with the highest plasmodesmatal counts have specialized companion cells known as intermediary cells (Fig 3) Intermediary cells are especially large, with many small vacuoles and extremely large num-bers of asymmetrically branched plasmodesmata connecting them to bundle sheath cells They are so different in many respects from the rest of the type plants that they should probably be treated as a separate group Type is also heterogeneous Type 2a companion cells have smooth cell walls, whereas those of type 2b have transfer cells with highly invaginated plasma membranes (Fig 3)

Plasmodesmatal frequency is often a strong family characteristic; for example, all studied spe-cies in the Magnoliaceae (magnolia family) are

type 1, those in the Aceraceae (maple family) are type 1-2a; and those in the Liliaceae (lily family) are type The minor veins of most monocots have low plasmodesmatal frequencies Trees tend to have more plasmodesmata in the loading pathway than herbaceous plants, but this is not a strict correlation, because both herbaceous and tree species are found in some families For example, Fragaria (strawberry) and Malus (apple) are both in the Rosaceae (rose family) and both are type 1-2a plants

3.3 Sugar Transport against a Concentration Gradient

As noted in Sect 1, one of the characteristics of the phloem is that the solute concentration, and thus the hydrostatic pressure, is high (Fig 4) How is this high solute concentration generated?In many spe-cies, sucrose is actively loaded into the phloem from the apoplast by specific transporters located in the plasma membranes of the companion cells and/or sieve elements(Fig 5A) By taking advantage of the steep proton gradient between the apoplast and the cytosol of the sieve elements, with pH values of approximately and 9, respectively, sucrose is con-tinually pumped into the phloem by secondary active transport, maintaining a concentration sev-eral times that found in mesophyll cells Since sucrose-proton co-transporters are found in the plasma membranes of most plant cells, it is

FIGURE Phloem transport Cell walls are

reasonable to assume that apoplastic phloem load-ingevolved from a general retrieval mechanism that returns to the cytoplasm sucrose that has leaked out of cells

If sucrose is loaded into the phloem from the apoplast, one would expect little symplastic conti-nuity with the mesophyll; otherwise the loaded sucrose would leak back through the plasmodes-mata to the cells it came from, creating a futile pump/leak system Indeed, all type plants (those with a low plasmodesmatal frequency) that have been studied load from the apoplast Many of these plants have highly invaginated plasma mem-branes (type 2b transfer cells) that maximize surface area for this transport What about the plants with intermediary cells (Fig 5A) that have numerous plasmodesmatabetween the mesophyll and minor vein phloem (Gamalei’s type and type 1—2a)? One possibility is that, in these species, sucrose simply diffuses along an entirely symplastic pathway from the mesophyll, without creating an uphill gradient into the phloem (Fig 5B) If so, the concentration of sucrose must be higher in the mesophyll than in the phloem This appears to be the case in Salix babylo-nica [(weeping willow), a type species with

numerous plasmodesmata], which has a high centration of sucrose in the leaves, but a lower con-centration in the phloem of the stem (Turgeon & Medville 1998)

In species with intermediary cells, yet another strategy prevails (Fig 5C) All plants with inter-mediary cells transport their photoassimilates primarily as raffinose and stachyose which sug-gests that the synthesis of these sugars (tri- and tetra-saccharides, respectively; Fig 2) is somehow part of the phloem-loading mechanism A model put forward to explain this is known as polymer trapping(Turgeon 1991, 1996) Sucrose supposedly diffuses into the intermediary cells from the bundle sheath through the numerous plasmodesmata that connect these two cell types Inside the intermediary cells, most of the sucrose is converted to raffinose and stachyose, which accumulate to high concentra-tions, because these sugars are too large to diffuse backward through the plasmodesmata This keeps the sucrose concentration lower in the intermediary cell than in the mesophyll, and allows continued diffusion Thus, the plasmodesmata between bun-dle sheath cells and intermediary cells act as valves The plasmodesmata between the intermediary cells

FIGURE Phloem-loading pathways and

mecha-nisms (A) Apoplastic loading Sucrose from mesophyll cells (M) diffuses through plasmodesmata to bundle sheath cells (BS) and into the minor veins Inside the veins, it enters the cell-wall space (apoplast, in grey) near the phloem, and is loaded into the companion cells (CC) and/or sieve elements (SE) by secondary active transport A sucrose transporter is shown as a star Phloem parenchyma cells (not shown) are part of the pathway in the vein, and may be the most impor-tant site of sucrose efflux into the apoplast Apoplastic loading is the most common strategy in flowering plants (B) Diffusion A downhill sucrose concentration gradient allows diffusion from the cytosol of mesophyll cells, through the bundle sheath and companion cells, and into the sieve elements Sucrose is carried away in the sieve tubes to the sinks, resulting in continued diffusion Phloem parenchyma cells are not shown here, but may also be part of the diffusion pathway in the vein (C) Polymer trapping Sucrose diffuses through numerous, narrow plasmodesmata from bun-dle sheath cells into specialized companion cells called intermediary cells (IC), where it is converted to raffi-nose (trisaccharide) and stachyose (tetrasaccharide) This keeps the sucrose concentration in the intermedi-ary cells low and prevents back diffusion to the meso-phyll The sugars pass from intermediary cells into the sieve elements through larger plasmodesmata Cour-tesy R Turgeon, Cornell University, Ithaca, U.S.A

and the sieve elements are larger, which permits entry of the sugars into the long-distance transport stream

Most effort in this field has been devoted to sucrose loading, since sucrose is the major transport compound in most plants We know less about the loading of sugar alcohols, in the species that trans-port them, and even less about the loading of other organic compounds and ions Unraveling these mechanisms is an ongoing research effort

4 Evolution and Ecology of Phloem Loading Mechanisms

The ancestral mechanism of phloem loading in flowering plants is not known for certain because we cannot be sure that ‘‘basal’’ groups (those that diverged early in the evolution of the angiosperms) have retained their ancestral characteristics (Fig 6) However, most basal plants have numerous minor vein plasmodesmata (type 1) (Gamalei 1989, Turgeon et al 2001) Type plants, and plants with intermediary cells, are more phylogenetically derived; these traits having evolved independently on a number of occasions (Turgeon et al 2001) The strategies employed by other vascular plants, including the gymnosperms, are not known

What are the selective pressures that have led to the emergence of the different forms of phloem loading? There is no clear answer to this question, but we can make a start by studying the growth characteristics and habitats of existing plants Families with intermediary cells are heavily repre-sented in the tropics [e.g., Cucurbitaceae (gourd family)] although a few are cosmopolitan [Scrophu-lariaceae (figwort family)] and some individual spe-cies occur in the arctic There appears to be no correlation of intermediary cells with growth rate or with the woody or herbaceous growth habit The rest of the type species are essentially all woody (trees or shrubs) and can be found in all climates, except the arctic Type plants can have many forms, but they tend to be herbaceous and are more heavily represented in temperate and colder regions

What the differences between phloem-load-ing types signify? An early hypothesis that symplas-tic loading is somehow more sensitive to the cold than apoplastic loading does not appear to be valid The absence of type plants in the arctic is probably due to the fact that there are very few woody species of any kind in those extremely cold environments, for reasons that have nothing to with phloem

loading In addition, laboratory experiments not support the concept of cold sensitivity in type species with intermediary cells (Schrier et al 2000) Although plants with intermediary cells seem to be favored in the tropics, it should not be assumed that this has anything directly to with temperature There are many other correlates of life in the tropics that need to be considered

Sugar alcohols present another problem Plants from many families produce sugar alcohols in their leaves, though only a few appear to transport sig-nificant amounts of these compounds in the phloem There is convincing evidence that sugar alcohols confer tolerance to boron deficiency because they complex and solubilize this otherwise insoluble mineral and allow it to be transported in the phloem from leaves to meristematic regions, where it is needed for growth (Hu et al 1997) It has also been suggested that sugar alcohol synthesis and export may channel away excess reducing energy from photosynthesis in times of stress (Loescher & Everard 2000)

5 Phloem Unloading

When considering how sugars and other materials unload from the phloem, it is useful to make the distinction between axial sinks (tissues adjacent to the axial, long-distance transport phloem in shoots and roots) and terminal sinks (tissues that are either actively growing or storing large quantities of photoassimilates, such as shoot and root tips, grow-ing leaves, and growgrow-ing fruits) (Fig 7)

FIGURE6 Minor vein companion cell characteristics for

137 taxa mapped onto a phylogenetic tree Wherever possible, genera for which phloem anatomy is known are coded directly in the matrix, but for some represen-tatives there is no equivalent genus in the molecular matrix In some of these cases, a closely related confa-milial genus is coded for the phloem character All taxa

scored as missing for the phloem loading character are automatically pruned from the consensus tree, produ-cing a tree topology that is a fully congruent subset of the topology with all taxa The tree has been split at the point indicated by the arrows, with the more ancestral taxa at the left (Turgeon et al 2001) Copyright The Botanical Society of America

surrounding the embryo The embryo then sca-venges the sucrose by active transporters in the plasma membranes of the cotyledons

Unloading in axial sinks follows either the sym-plasticor apoplastic routes, depending on the spe-cies, the specific sink, and the stage of development When the path of small fluorescent dyes is followed down a root, the dye does not exit the axial phloem, indicating that the plasmodesmata are too narrow to accommodate even small solute molecules (Oparka et al 1994) Therefore, sugars and other solutes must be released into the apoplast However, as indicated above, when the dye in the phloem reaches the meristematic tissue at the tip of the root, it rapidly unloads through plasmodesmata

Unloading into cotton fibers provides an exam-ple of a shift in unloading routes during develop-ment Cotton fibers (single cells of the seed coat epidermis) grow extremely rapidly Initially, unloading from the phloem and post-phloem trans-port into the fiber cell is entirely symplastic How-ever, during the most rapid growth phase the plasmodesmata in the wall of the fiber close for about days, and sucrose instead enters the apo-plast This allows active sucrose transporters in the plasma membrane of the fiber to drive up osmotic and turgor potentials to the high values needed for rapid cell expansion (Ruan et al 2001) Once this active growth phase is over, the plasmodesmata open again

FIGURE Phloem unloading, followed by transport into

developing seeds The diagram shows membrane trans-porters involved in transferring phloem-imported nutri-ents from seed coats to cotyledons of developing grain legume seeds Phloem-imported nutrients are trans-ported through plasmodesmata to the seed coat efflux cells Here, membrane transporters in plasma mem-branes lining the efflux cells release nutrients to the seed apoplast Currently known transporters are: (1) nonselective channels; (2) sucrose/H+antiporters;

(3) H+-ATPases; (4) sucrose facilitators; (5) aquaporins;

(6) sucrose/H+ symporters; (7) pulsing Cl- channels.

Nutrients are taken up from the seed apoplast by

membrane transporters located in plasma membranes of cotyledon cell complexes Currently known transpor-ters include: (8) nonselective cation channels; (9) sucrose/H+ symporters; (10) H+-ATPases; (11) amino

acid/H+ symporters; (12) hexose/H+ symporters An

elevated cell turgor (arrow), due to enhanced uptake of nutrients from the seed apoplast, activates Cl-and

nonselective channels, and possibly also activates Ca2+

release, leading to an increase in the cytosolic Ca2+

Phloem unloading in sink leaves illustrates the need to consider anatomy, physiology, and devel-opment to assemble a complete picture of events (Turgeon 2006) Very young leaves are sinks: they obtain most of their carbohydrate from older leaves As this photoassimilate enters a young leaf in the phloem it unloads from relatively large veins; the smaller veins are not yet mature (Turgeon 1987, Roberts et al 1997) As the leaf grows, it reaches a positive carbon balance and then begins to export Just before it does so, the small veins mature These minor veinsare used for photoassimilate loading Therefore, there is a division of labor between veins of different size classes, large ones for unloading in young leaves, small ones for loading in mature leaves

In some organisms structures have evolved to parasitize the phloem-transport system Rapid phloem unloading occurs when a phloem-feeding organism (e.g., an aphid) injects its stylet into a sieve tube The hydrostatic pressure in the sieve pushes the contents of the sieve tube into the aphid The aphid absorbs predominantly nitrogen-ous compounds and excretes much of the carbohy-drate as ‘‘honeydew’’ The aphids ingest phloem sap without eliciting the sieve tubes’ normal response to injury (Sect 3) Sealing mechanisms are prevented by chemical constituents in aphid saliva injected into sieve tubes before and during feeding (Will et al 2007) Another special site where phloem unloading occurs is the haustoria ofholoparasites that depend on their host for their carbon supply The release of solutes from the phloem of the host is strongly stimulated by the presence of such a parasite, by an as yet unidenti-fied mechanism (Sect of Chapter 9D on parasitic associations) In some species, e.g., Lupinus albus (white lupin) the phloem bleeds spontaneously upon cutting (Pate & Hocking 1978) Phloem sap collected in this way or as honeydew has provided valuable information on the composition of phloem sap (Sect 2)

Phloem unloading is affected in a rather special manner byroot nematodes (e.g., the parasitic nema-todes Meloidogyne incognita and Heterodera schachtii), which can act as major sinks (Dorhout et al 1993) Unloading from the sieve element companion cell complexes occurs specifically into the ‘‘syncytium’’, the nematode-induced feeding structure within the vascular cylinder of the root The infective juvenile nematode selects a procambial or cambial cell as an initial syncytial cell, from which a syncytium ops by integration of neighboring cells The devel-oping nematode depends entirely on the expanding syncytium, withdrawing nutrients from it through a

feeding tube Unlike in the root tip, the transport of sugars from the phloem to the syncytium in this host-pathogen relationship is apoplastic The syncy-tium is not connected via plasmodesmata with the normal root cells In an as yet unidentified manner, the nematode induces massive leakage from the phloem, thus reducing the transport of phloem solutes to the rest of the roots (B ăockenhoff et al 1996)

6 The Transport Problems of Climbing Plants

Vines can be viewed as ‘‘mechanical parasites’’ They invest too little in wood to support them-selves, and thus depend on other plants for mechanical support Xylem (wood) tissue has both a transport and a mechanical support func-tion As discussed in Sect 5.3.5 of Chapter on plant water relations, vines have fewer but longer and wider xylem vessels in their stem per unit stem cross-sectional area They also have fewer lignified phloem fibers than trees and shrubs and less phloem tissue per unit of distal area (Fig 8) How vines achieve sufficient phloem transport capacity?

Compared with trees and shrubs, vines have wider sieve tubes (Fig 8) Since the hydraulic conductance, by Hagen-Poiseuille’s law for ideal capillaries, is proportional to the fourth power of the conduit radius (Sect 5.3.1 of Chapter on plant water relations), the larger diameter compensates for the smaller total area The obvious advantage of fewer sieve tubes with a larger diameter is that relatively few resources need to be allocated to producing phloem in the stem, which is therefore light, preventing the supporting plant from top-pling over For a similar investment in stem, the climbing plant will reach a greater height than a nonclimbing plant If few sieve tubes with large diameters are so advantageous for climbing plants, why not all plants have such wide tubes in their phloem? There is likely a disadvantage in having large-diameter sieve tubes, in that physical damage to a small number of sieve tubes causes a larger proportional loss of transport capacity Such damage may be mechanical or due to phloem-sucking arthropods or pathogens As in the xylem of plants with contrasting strategy (Sect 5.3.5 of Chapter on plant water relations), there may be a trade-off between transport capacity and safety

7 Phloem Transport: Where to Move from Here?

After several years of debate on whether phloem loading occurs via an apoplastic or a symplastic pathway, it is now agreed that both pathways occur, depending on species Are there disadvan-tages and disadvandisadvan-tages associated with the apo-plastic or symapo-plastic pathway? The proton-pumping activity of the transfer cells involved in apoplastic loading requires a substantial amount of metabolic energy It remains to be demon-strated, however, that this energy requirement is greater than that for the polymerization that occurs in the intermediary cells of plants with symplastic phloem loading Disadvantages asso-ciated with the apoplastic pathway are not imme-diately obvious

Phloem unloading can also occur either apoplas-tically or symplasapoplas-tically, depending on the kind of sink and on species Phloem unloading in sinks is an important aspect of crop yield, since increases in

yield in newer varieties are often determined by the amount of resources transported to harvestable sinks, rather than by the total amount of resources acquired It is therefore important to develop a good understanding of phloem transport Unraveling both loading and unloading mechanisms continues to offer major challenges

References

B ăockenhoff, A., Prior, D.A.M., Gruddler, F.M.W., & Oparka, K.J 1996 Induction of phloem unloading in Arabidopsis thaliana roots by the parasitic nema-tode Heterodera schachtii Plant Physiol 112: 1421—1427

Dorhout, R., Gommers, F.J., & Koll ăoffel, C 1993 Phloem transport of carboxyfluorescein through tomato roots infected with Meloidogyne incognita Physiol Mol Plant Pathol 43: 1—10

Ewers, F.W & Fisher, J.B 1991 Why vines have narrow stems: Histological trends in Bauhinia fassoglensis (Faba-ceae) Oecologia 88: 233—237

FIGURE Phloem area (A) and maximum diameters

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3

Plant Water Relations

1 Introduction

Although water is the most abundant molecule on the Earth’s surface, the availability of water is the factor that most strongly restricts terrestrial plant production on a global scale Low water availability limits the productivity of many natural ecosystems, particularly in dry climates (Fig 1) In addition, losses in crop yield due to water stress exceed losses due to all other biotic and environmental factors combined (Boyer 1985) Regions where rainfall is abundant and fairly evenly distributed over the growing season, such as in the wet tropics, have lush vegetation Where summer droughts are frequent and severe, forests are replaced by grass-lands, as in the Asian steppes and North American prairies Further decrease in rainfall results in semide-sert, with scattered shrubs, and finally deserts Even the effects of temperature are partly exerted through water relations because rates of evaporation and tran-spiration are correlated with temperature Thus, if we want to explain natural patterns of productivity or to increase productivity of agriculture or forestry, it is crucial that we understand the controls over plant water relations and the consequences for plant growth of an inadequate water supply

1.1 The Role of Water in Plant Functioning

Water is important to the physiology of plants because of its crucial role in all physiological

processes and because of the large quantities that are required Water typically comprises 70—95% of the biomass of nonwoody tissues such as leaves and roots At the cellular level, water is the major med-ium for transporting metabolites through the cell Because of its highly polar structure, water readily dissolves large quantities of ions and polar organic metabolites like sugars, amino acids, and proteins that are critical to metabolism and life At the whole-plant level, water is the medium that transports the raw materials (carbohydrates and nutrients) as well as the phytohormones that are required for growth and development from one plant organ to another Unlike most animals, plants lack a well-developed skeletal system; especially herbaceous plants depend largely on water for their overall structure and support Due to their high concentra-tions of solutes, plant cells exert a positive pressure (turgor) against their cell walls, which is the basic support mechanism in plants Turgor pressures are typically of the order of 1.0—5.0 MPa, similar to the pressure in nuclear steam turbines Large plants gain additional structural support from the lignified cell walls of woody tissues When plants lose turgor (wilt), they no longer carry out certain physiological functions, in particular cell expansion and to a lesser extent photosynthesis Prolonged periods of wilting usually kill the plant

A second general reason for the importance of water relations to the physiological ecology of plants is that plants require vast quantities of water Whereas plants incorporate more than 90% of

H Lambers et al., Plant Physiological Ecology, Second edition, DOI: 10.1007/978-0-387-78341-3_3,  Springer ScienceỵBusiness Media, LLC 2008

absorbed N, P, and K, and about 10—70% of photo-synthetically fixed C into new tissues (depending on respiratory demands for carbon), less than 1% of the water absorbed by plants is retained in biomass (Table 1) The remainder is lost by transpiration, which is the evaporation of water from plants The inefficient use of water by terrestrial plants is an unavoidable consequence of photosynthesis The sto-mates, which allow CO2to enter the leaf, also provide a pathway for water loss CO2that enters the leaf must first dissolve in water on the wet walls of the mesophyll cells before diffusing to the site of carbox-ylation This moist surface area of mesophyll cells exposed to the internal air spaces of the leaf is about 7—80 times the external leaf area, depending on spe-cies and plant growth conditions (Table 2) This causes the air inside the leaf to be saturated with water vapor (almost 100% relative humidity) which

creates a strong gradient in water vapor concentration from the inside to the outside of the leaf

1.2 Transpiration as an Inevitable Consequence of Photosynthesis

Transpiration is an inevitable consequence of photo-synthesis; however, it also has important direct effects on the plant because it is a major component of the leaf’s energy balance As water evaporates from mesophyll cell surfaces, it cools the leaf In the absence of transpiration, the temperature of large leaves can rapidly rise to lethal levels We TABLE Concentration of major constituents in a

hypothetical herbaceous plant and the amount of each constituent that must be absorbed to produce a gram of dry biomass The values only give a rough approximation and vary widely among species and with growing conditions, indicated in Sect 4.3 of Chapter on mineral nutrition for nutrients, and in Sect for water

Resource

Concentration (% of fresh mass)

Quantity required (mg g–1)

Water 90 500,000

Carbon 40

Nitrogen 0.3

Potassium 0.2

Phosphorus 0.02 0.2

FIGURE Correlation of

above-ground net primary pro-duction (NPP, in units of bio-mass) with precipitation NPP declines at extremely high pre-cipitation (>3 m yr–1) due to indirect effects of excess moisture, such as low soil oxy-gen and nutrient loss by leach-ing (Schuur 2003) Copyright Ecological Society of America

TABLE The ratio of the surface area of mesophyll cells and that of the leaf (Ames/ A) as dependent on species and growing conditions.*

Leaf morphology/habitat Ames/A

Shade leaves

Mesomorphic leaves 12–19 Xeromorphic sun leaves 17–31 Low altitude (600 m) 37 High altitude (3000 m) 47

Species Ames/A

Plectranthus parviflorus

High light 39

Low light 11

Alternanthera philoxeroides

High light 78

Low light 50

*

The data on leaves of species with different morphologies are from Turrel (1936), those on low-altitude and high-altitude species from Kăorner et al (1989), those on Plectranthus parvi-florus from Nobel et al (1975), and those on Alternanthera philoxeroides (alligator weed) from Longstreth et al (1985)

further discuss this effect of transpiration in Chapter 4A on the plant’s energy balance The transpiration stream also allows transport of nutrients from the bulk soil to the root surface and of solutes, such as inorganic nutrients, amino acids, and phytohor-mones, from the root to transpiring organs As will be discussed later, however, such transport in the xylem also occurs in the absence of transpiration, so that the movement of materials in the transpiration stream is not strongly affected by transpiration rate In this chapter, we describe the environmental factors that govern water availability and loss, the movement of water into and through the plant, and the physiological adjustments that plants make to variation in water supply over diverse timescales We emphasize the mechanisms by which individual plants adjust water relations in response to variation in water supply and the adaptations that have evolved in dry environments

2 Water Potential

The status of water in soils, plants, and the atmo-sphere is commonly described in terms of water potential( w) [i.e., the chemical potential of water in a specified part of the system, compared with the chemical potential of pure water at the same tem-perature and atmospheric pressure; it is measured in units of pressure (MPa)] The water potential of pure, free water at atmospheric pressure and at a tempera-ture of 298 K is MPa (by definition) (Box 3.1)

In an isothermal two-compartment system, in which the two compartments are separated by a semipermeable membrane, water will move from a high to a low water potential If we know the water potential in the two compartments, then we can predict the direction of water movement It is cer-tainly not true, however, that water invariably moves down a gradient in water potential For example, in the phloem of a source leaf, the water potential is typically more negative than it is in the phloem of the sink In this case, water transport is driven by a difference in hydrostatic pressure, and water moves up a gradient in water potential Simi-larly, when dealing with a nonisothermal system, such as a warm atmosphere and a cold leaf, water vapor may condense on the leaf even though the water potential of the air is more negative than that of the leaf

Water potential in any part of the system is the algebraic sum of the osmotic potential, p, and the hydrostatic pressure, p(the component of the water potential determined by gravity is mostly ignored):

wẳ pỵ p (1)

where water potential is the overall pressure on water in the system The osmotic potential is the chemical potential of water in a solution due to the presence of dissolved materials The osmotic poten-tial always has a negative value because water tends to move across a semipermeable membrane from pure water (the standard against which water potential is defined) into water containing solutes (Box 3.1) The higher the concentration of solutes, the lower (more negative) is the osmotic potential The hydrostatic pressure, which can be positive or negative, refers to the physical pressure exerted on water in the system For example, water in the tur-gid root cortical cells or leaf mesophyll cells is under positive turgor pressure exerted against the cell walls, whereas water in the dead xylem vessels of a rapidly transpiring plant is typically under suc-tion tension (negative pressure) Large negative hydrostatic pressures arise because of capillary effects, i.e., the attraction between water and hydro-philic surfaces at an air—water interface (Box 3.2) Total water potential can have a positive or negative value, depending on the algebraic sum of its com-ponents When dealing with the water potential in soils, an additional term is used: the matric poten-tial, m The matric potential refers to the force with which water is adsorbed onto surfaces such as cell walls, soil particles, or colloids, similar to the forces in xylem vessels As such it is actually a convenient alternativeto hydrostatic pressure for characterizing the water status of a porous solid The hydrostatic pressure and the matric potential should therefore never be added! The matric potential always has a negative value because the forces tend to hold water in place, relative to pure water in the absence of adsorptive surfaces The matric potential becomes more negative as the water film becomes thinner (smaller cells or thinner water film in soil)

Now that we have defined the components of water potential, we show how these components vary along the gradient from soil to plant to atmosphere

3 Water Availability in Soil

Box 3.1

The Water Potential

of Osmotic Solutes and the Air

We are quite familiar with the fact that water can have a potential: we know that water at the top of a falls or in a tap has a higher potential than that at the bottom of the falls or outside the tap Transport of water, however, occurs not invari-ably as a result of differences in hydrostatic pressure, but also due to differences in vapor pressure (Sect 2.2.2 of Chapter 2A on photo-synthesis) or due to differences in the amount of dissolved osmotic solutes in two compart-ments separated by a semipermeable membrane In fact, in all these cases, there is a difference in water potential, which drives the transport of water For a full appreciation of many aspects of plant water relations, we first introduce the con-cept of the chemical potential of water, for which we use the symbol mw

By definition, the chemical potential of pure water under standard conditions (298 K and standard pressure), for which the symbol 0

w is used, is zero We can also calculate the chemical potential of water under pressure, of water that contains osmotic solutes, or of water in air This can best be explained using a simple example, comparing the chemical potential of water in two sealed containers of similar size (Fig 1) One of these containers (A) contains pure water under standard conditions: w¼ ow¼ Of course, the gas phase is in equilibrium with the liquid pure water, and the vapor pressure is po The second container (B) contains a M sucrose solution in water The gas phase will again be in equilibrium with the liquid phase; the vapor pressure is p The vapor pressure, however, will be less than po because the sucrose molecules interact with the water molecules via hydrogen bonds, so that the water molecules cannot move into the gas phase as readily as in the situation of pure water How large is the difference between p and po?

To answer this question, we use Raoult’s law, which states that

p=p0¼ Nw (1)

where Nwis the mol fraction [i.e., the number of moles of water divided by the total number of moles in container B; in the case of mole of sucrose in L water (55.6 moles of water), Nw=

55.6/56.6 = 0.982]; pois the vapor pressure (in Pa) above pure water, at standard pressure and tem-perature We can calculate the difference in poten-tial between the two containers ðw owÞ by considering the amount of work needed to obtain the same (higher) pressure in container B as in container A To achieve this, we need to compress the gas in container B until the pressure equals po:

w 0w¼ Zp

p0

V dp ¼ RT ln p p0  

(2)

where V is the volume (m3) of container B, which is compressed until pois reached, R is the gas constant (J mol—1K—1), and T is the absolute temperature (K)

Combination of Equations (1) and (2) yields

w ow¼ RT lnð1  NwÞ (3)

Because Nwis the mole fraction of water and Nsis the mole fraction of the solute (in our exam-ple, 1/56.6 = 0.018), we can write Equation (3) as

w owẳ RT ln1  Nsị (4)

As long as we consider solutions in a physio-logically relevant range (i.e., not exceeding a few molar) Equation (4) approximates

w ow¼ RT Ns (5)

[as can readily be calculated for our example of a M solution of sucrose, Ns is 0.018 and ln(1  Ns) = —0.018]

Dividing Ns by the molar volume of pure water (Vwom3mol—1), we arrive at the concentra-tion of the solute, cs(in mol m—3):

Ns=Vow¼ cs (6)

We make one further change, by introducing the molar volume of pure water (m3 mol—1; at 273 K) in Equation (5):

w ow Vwo

¼ RTcs ¼  (7)

continued

Box 3.1.Continued

is the water potential Because we are deal-ing with the water potential of a solution in this example, we refer to this potential as the osmotic potential of waterðpÞ The dimension is Pascal (Pa) It is often more convenient, however, to use megapascal (MPa = 106Pa) instead (1 MPa = 10 bars, a unit used in the literature, or 10 atm, a unit that is no longer used)

We can therefore calculate that our M sucrose solution has an osmotic potential of —2.4 MPa, which approximates a pressure of a water column of about 250 m! In equilibrium, the water potential of the gas phase above the M sucrose solution also equals —2.4 MPa In the case of electrolytes, the calculation is slightly more complicated in that the dissociation of the solute has to be taken into account

By modifying Equation (7), we can also calculate the water potential of air that is not in equilibrium with pure water [i.e., with a relative humidity (RH) of less than 100%]:

w ow Vo

w

ẳRT Vo w

lnp po

ị (8)

For air of 293 K and a RH of 75%,  equals —39 MPa [to calculate this, you need to know that the molar volume of water (molecular mass = 18) at 293 K is 18.10—6m3mol—1] Values for  of air of different RH are presented in Table Note that

even when the water vapor pressure is only mar-ginally lower than the saturated water vapor pres-sure RH = 100% , the water potential is rather negative

TABLE The water potential (MPa) of air at a range of relative humidities and temperatures *

Relative humidity

(%) -Y (MPa) at different temperatures (8C)

10 15 20 25 30

100 0 0

99.5 0.65 0.67 0.68 0.69 0.70 99 1.31 1.33 1.36 1.38 1.40 98 2.64 2.68 2.73 2.77 2.81 95 6.69 6.81 6.92 7.04 7.14 90 13.75 13.99 14.22 14.45 14.66 80 29.13 29.63 30.11 30.61 31.06 70 46.56 47.36 48.14 48.94 49.65 50 90.50 92.04 93.55 95.11 96.50 30 157.2 159.9 162.5 165.2 167.6 10 300.6 305.8 310.8 316.0 320.6 RT/Vw 130.6 132.8 135.0 137.3 139.2

Note: The values were calculated using the formula: Y = –RT/Vw0ln (% relative humidity/100)

* Note that all values for Y are negative and that the effect of temperature is exclusively due to the appearance of temperature in the equation given in the last line of this table, rather than to any effect of temperature on po

FIGURE1 The difference in water potential between

two systems The system at the left is a sealed con-tainer with pure water at standard temperature and pressure; the partial water vapor pressure in this container is poand the chemical potential of water

in this system is mwo The system at the right is a

container with a solution of M sucrose at the

same temperature and pressure; the water vapor pressure can be calculated according to Raoult’s law (p = po.Nw) and the chemical potential of water

in this system is mw The difference in chemical

(Box 3.2) Pores larger than 30 mm hold the water only rather loosely, so the water drains out follow-ing a rain Pores smaller than 0.2 mm hold water so tightly to surrounding soil particles that the drainage rate often becomes very small once the large pores have been drained As a result, most plants cannot extract water from these pores at sufficiently high rates to meet their water needs It is thus the intermediate–sized pores (0.2—30 mm diameter) that hold most of the water that is tapped by plants

In friable soil, roots can explore a large fraction of the soil volume; hence, the volume of water that is available to the roots is relatively large Upon soil compaction, roots are unable to explore as large a fraction of the soil volume; the roots then tend to be clumped into sparse pores and water uptake is restricted Compacted soils, however, are not uni-formly hard and usually contain structural cracks and biopores (i.e., continuous large pores formed by soil fauna and roots) Roots grow best in soil with an intermediate density, which is soft enough to allow

Box 3.2

Positive and Negative Hydrostatic Pressures

Positive values of hydrostatic pressure in plants are typically found in living cells and are accounted for by high concentrations of osmotic solutes Large negative values arise because of capillary effects (i.e., the attraction between water and hydrophilic surfaces at an air—water interface) It is this attraction that explains the negative matric potential in soil and the negative hydrostatic pres-sure in the xylem of a transpiring plant

The impact of the attraction between water and hydrophilic surfaces on the pressure in the adjacent water can be understood by imagin-ing a glass capillary tube, with radius a (m), placed vertically with one end immersed in water Water will rise in the tube, against the gravitational force, until the mass of the water in the tube equals the force of attraction between the water and the glass wall A fully developed meniscus will exist (i.e., one with a radius of curvature equal to that of the tube) The menis-cus of the water in the glass tube is curved because it supports the mass of the water

The upward acting force in the water column equals the perimeter of contact between water and glass (2pa) multiplied by the surface tension, ðN m1Þ, of water; namely, 2pag (provided the glass is perfectly hydrophilic, when the contact angle between the glass and the water is zero; otherwise, this expression has to be multiplied by the cosine of the angle of contact) When in equilibrium, there must be a difference in pres-sure, P (Pa) across the meniscus, equal to the force of attraction between the water and the capillary wall (i.e., the pressure in the water is less than that of the air) The downward acting

force (N) on the meniscus is the difference in pressure multiplied by the cross-sectional area of the capillary tube (i.e., pa2gP) Thus, because these forces are equal in equilibrium, we have

pa2P ¼2pa (1)

and

P ¼2pa=pa2¼ 2=a (2)

The surface tension of water is 0.075 N m—1at about 208C, so P = 0.15/a (Pa) Thus a fully developed meniscus in a cylindrical pore of radius, say 1:5 m, would have a pressure drop across it of 1.0 MPa; the pressure, P, in the water would therefore be —0.1 MPa if refer-enced to normal atmospheric pressure, or —0.9 MPa absolute pressure (given that standard atmospheric pressure is approximately 0.1 MPa)

This reasoning also pertains to pores that are not cylindrical It is the radius of curvature of the meniscus that determines the pressure difference across the meniscus, and this curva-ture is uniform over a meniscus that occupies a pore of any arbitrary shape It is such capillary action that generates the large negative pressures (large suction tension) in the cell walls of leaves that drive the long-distance transport of water from the soil through a plant to sites of evapora-tion The pores in cell walls are especially small (approximately nm) and are therefore able to develop very large suction tensions, as they in severely water-stressed plants

good root growth but sufficiently compact to give good root—soil contact (Stirzaker et al 1996)

Water movement between root and soil can be limited by incomplete root—soil contact, such as that caused by air gaps due to root shrinkage during drought It can also be influenced by a rhizosheath (i.e., the soil particles bound together by root exu-dates and root hairs) (McCully & Canny 1988) Rhizosheaths are limited to distal root regions, which generally have a higher water content than the more proximal regions (Huang et al 1993) in part due to the immaturity of the xylem in the distal region (Wang et al 1991) The rhizosheath virtually eliminates root—soil air gaps, thus facilitating water uptake in moist soil On the other hand, bare roots restrict water loss from roots to a drier soil (North & Nobel 1997)

3.1 The Field Capacity of Different Soils

Field capacityis defined as the water content after the soil becomes saturated, followed by complete gravitational drainage The water potential of non-saline soils at field capacity is close to zero (—0.01 to —0.03 MPa) There is a higher soil water content at field capacity in fine-textured soils with a high clay

or organic matter content (Fig 2) The lowest water potential at which a plant can access water from soil is the permanent wilting point Although species differ in the extent to which they can draw down soil water (e.g., from —1.0 to —8.0 MPa), as discussed later, a permanent wilting point of —1.5 MPa is com-mon for many herbaceous species The available wateris the difference in the amount of soil water between field capacity and permanent wilting point, —1.5 MPa (by definition) The amount of available water is higher in clay than it is in sandy soils (Fig 2, Table 3)

In a moist soil, the smallest soil pores are comple-tely filled with water and only the largest pores have air spaces As soil moisture declines, the thickness of the water film surrounding soil particles declines, and remaining water is held more tightly to soil particles, giving a low (negative) matric potential Finally, the hydrostatic pressure (reflecting gravity or the mass of the water column) is generally negligible in soils In nonsaline soils, the matric potential is the most impor-tant component of soil water potential

In saline soils, the osmotic potential adds an additional important component If plants are well watered with a saline solution of 100 mM NaCl, then the soil water potential is —0.48 MPa As the soil dries out, the salts become more concentrated and further add to the negative value of the soil water potential When half of the water available at field capacity has been absorbed, the osmotic component of the soil water potential will have dropped to almost —1 MPa Under such situations, the osmotic component of the soil water potential, clearly, can-not be ignored

Soil organic matter affects water retention because of its hydrophilic character and its influence on soil structure Increasing the organic matter con-tent from 0.2 to 5.4% more than doubles the water-holding capacity of a sandy soil—from 0.05 to 0.12

FIGURE2 Relationship between soil water potential and

volumetric soil water content (ratio of volume taken up by water and total soil volume, ) at different soil depths: 25 cm, solid triangles; 50–80 cm, open circles; 110–140 cm, filled circles The top horizon was a silty clay loam; the middle layer was enriched with clay, and in the deepest soil layer, the clay content decreased again Soil water potential was measured with tensi-ometers and micropsychrtensi-ometers, and soil water con-tent with a neutron probe Data were obtained over year while water content fell during drought (Bre´da et al 1995)

TABLE3 Typical pore-size distribution and soil water contents of different soil types

Soil type

Sand Loam Clay

Parameter

Pore space (% of total)

>30 m particles 75 18

0.2–30 m 22 48 40

<0.2 m 34 53

Water content (% of volume)

(v/v) In silty soils, which have a larger water-holding capacity, the absolute effect of organic matter is similar, but less dramatic when expressed as a per-centage; it increases from about 0.20 to less than 0.30 (v/v) Effects on plant-available water content are smaller because the water content at field capacity as well as that at the permanent wilting point is enhanced (Kern 1995) Roots, especially mycorrhizal roots (Sect 2.5 of Chapter 9A on symbiotic associa-tions), may promote the development of soil aggre-gates, through the release of organic matter, and thus affect soil hydraulic properties Organic matter may also have the effect of repelling water, if it is highly hydrophobic Such situations may arise when plant-derived waxy compounds accumulate on the soil surface These reduce the rate at which water penetrates the soil so that much of the preci-pitation from a small shower may be lost through runoff or evaporation rather than becoming avail-able for the plant Some roots release surfactants, which may counteract the effect of water-repelling compounds in soil (Read et al 2003)

3.2 Water Movement Toward the Roots

Water moves relatively easily through soil to the roots of a transpiring plant by flowing down a gradient in hydrostatic pressure If the soil is especially dry (with a water potential less than —1.5 MPa), then there may be significant movement as water vapor Under those conditions, however, transpiration rates are very low Gradients in osmo-tic potential move little water because the transport coefficients for diffusion are typically orders of magnitude smaller than for flow down a hydro-static gradient Movement across the interface between root and soil is more complicated There may be a mucilaginous layer that contains pores so small that the flow of water across it is greatly hindered There may also be a lack of hydraulic

continuity between root and soil if the root is grow-ing in a pore wider than itself or if the root has shrunk A root has, generally, access to all available water within mm of the root As the soil dries and the matric forces holding water to soil particles increases, movement of liquid water through soils declines (Fig 3)

In a situation where the soil is relatively dry and the flow of water through it limits water uptake by the roots, the following equation approximates water uptake by the roots:

d0=dt ẳ D0 aị=2b2 (2)

where d0/dt is the rate of fall of mean soil water content, 0, with time, t; D is the diffusivity of soil water, which is approximately constant with a value of  10—4m2day—1(0.2  10—8m2s—1), during the extraction of about the last third of the available water in the soil (Fig 3), when the flow is likely limiting the rate of water uptake; a is the soil water content at the surface of the root; and b is the radius of a putative cylinder of soil surrounding the root, to which that root effectively has sole access, and can be calculated as b = (pL)—1/2, where L (m m—3), the rooting density, is the length of root per unit volume of soil (m3) (Passioura 1991).

Under the reasonable assumption that ais con-stant, as it would be if the root were maintaining a constant water potential of, say, —1.5 MPa at its sur-face (Fig 3), the equation can be integrated to give

0aịdẳ 0

aị0expDt=2b2ịẳd0expt=tị (3)

where (’ — a)0is (’ — a) when t = 0, and t*(equal to 2b2/D) is the time constant for the system: the time taken for the mean soil water content to fall to 1/e (i.e., 0.37) of its initial value If D is  10—4m2day—1, then t* is simply b2  10—4 days If the roots are evenly distributed in the soil, then, even at a low rooting density, L, of 0.1 m m—3, t*(calculated from

FIGURE3 The matric potential and diffusivity of soil water as a function of the volumetric water content (ratio of volume taken up by water and total soil volume) of a sandy loam soil (55% coarse sand, 19% fine sand, 12% silt, and 14% clay) (after Stirzaker & Passioura 1996)

b2= 1/[pL)]) is only about days Roots, therefore, should readily be able to extract all the available water from the soil When the soil is compacted, roots are not distributed so evenly through the soil (Sect 5.5 of Chapter on growth and allocation), and Equations (2) and (3) are no longer applicable Under those conditions, t*could become of the order of weeks The parameter t*changes with soil type and soil depth, but is not strongly affected by the nature of the plant extracting the water (Passioura 1991)

If a plant does not absorb all the ions arriving at the surface of its roots, the osmotic potential will drop locally, either only in the apoplast of the roots or possibly in the rhizosphere as well This is more pronounced in fertilized or saline soils than in nutri-ent-poor, nonsaline soils The effect is that plants have greater difficulty in extracting water from soil than expected from the average soil water potential (Stirzaker & Passioura 1996)

3.3 Rooting Profiles as Dependent on Soil Moisture Content

As long as the upper soil is fairly moist, plants tend to absorb most of their water from shallower soil regions, which is where roots are concentrated As the soil dries out, relatively more water is absorbed from deeper layers Water from the deepest layers, even from those where no roots penetrate, may become available through capillary rise (Fig 4; Bre´da et al 1995) The actual rooting depth varies greatly among species, with some chaparral shrubs

[Adenostoma fasciculatum (chamise), Quercus dumosa (California scrub oak), and Quercus chrysolepis (can-yon live oak)] growing in the San Gabriel and San Bernardino mountains in southern California reaching depths of m in fractured rock structures (Hellmers et al 1955) Maximum rooting depths are found in deserts and tropical grasslands and savan-nas (Canadell et al 1996) On the Edwards Plateau of central Texas, United States, rooting depths of a range of species were determined by using DNA sequence variation to identify roots from caves to 65 m deep At least six tree species in the system produced roots deeper than m, but only the ever-green oak, Quercus fusiformis, was found below 10 m The maximum rooting depth for the ecosystem was approximately 25 m (Jackson et al 1999) In the Kalahari Desert, well drillers must bore to great depths in very dry sand to reach water, and drillers reported some of the deepest roots thus far recorded in the world at 68 m In the Kalahari sands, the annual precipitation of less than 300 mm can only penetrate a couple of meters at most Below this wetting front, roots must grow in very dry sand for tens of meters before they can reach deep geologic water (Jennings 1974, as cited in Schulze et al 1988) A potential mechanism that would facilitate this growth in very dry sand is through hydraulic redis-tribution (Sect 5.2; Schulze et al 1988)

The root-trench method, in combination with measurements of volumetric soil water content (Fig 4), is a laborious and expensive method to obtain information on where most of the water comes from that a tree transpires If the isotope signatureof water differs among soil layers, then

FIGURE (Left) Rooting profile of Quercus petraea

(sessile oak) as dependent on soil depth Roots are divided in different diameter classes (Right) Volu-metric water content of the soil in which the oak tree was growing, as dependent on depth and time

this value can be used to obtain information on which soil layers and associated roots provide the water that is transpired (Box 3.3) This technique has shown that perennial groundwater sources can be important (Thorburn & Ehleringer 1995, Boutton et al 1999) For example, in a Utah desert scrub

community, most plants use a water source derived from winter storm recharge for their early spring growth (Ehleringer et al 1991) As this water source is depleted, however, only the deep-rooted woody perennials continue to tap this source, and more shallow-rooted species such as annuals, herbaceous

Box 3.3

Oxygen and Hydrogen Stable Isotopes

Small fractions of the elements H and O occur as their heavy stable isotopes 2H (also called deuterium; D) and 18O (0.156 and 1.2%, respectively) Their abundances in water (and CO2) in the immediate environment of the plant, and in water, metabolites, and macro-molecules in the plant itself vary as a result of fractionation processes operating in these two compartments Isotopic composition, which can be measured with high precision, pro-vides information about environmental and physiological parameters that is otherwise dif-ficult to obtain Isotopes in xylem water, for instance, can yield information on the source of water tapped by a plant, and isotopes in leaf water are influenced by stomatal conduc-tance and humidity Isotopes in plant dry matter can give a integrated and time-resolved historical record of environmental and physiological processes, as in tree rings (Dawson et al 2002) A problem, however, with interpreting isotopic composition of plant dry matter or of specific compounds (e.g., cellulose) in field studies is that it is simultaneously influenced by many factors Models have been developed to resolve these problems as much as possible (Farquhar et al 1998, Roden et al 2000, Gessler et al 2007)

Isotopes are measured as atomic ratios (R = rare isotope/common isotope) using mass spec-trometers and are expressed relative to a stan-dard (Stanstan-dard Mean Ocean Water; SMOW; see also Box 2A.2 and Box 2B.1):

2H or 18O%

oịẳRsample=Rstandard1ị1000 (1)

The d2H and d18O of precipitation water and other water bodies that are regularly involved in the global water cycle (meteoric waters) vary as a result of fractionation during evaporation and condensation in a temperature-dependent man-ner Tropical regions are characterized by d-values close to ocean water, and these d-values decrease toward the poles, particularly in winter

Depleted values are also found at higher alti-tudes and further inland on continents Fractio-nation processes in meteoric water operates similarly for 2H and18O, and the d-values are linearly related This is known as the global meteoric water line [d18O = (d2H — 10)/8] Frac-tionation processes in more closed compart-ments result in deviations from this line

Soil moisture in surface layers is typically iso-topically enriched as a result of evaporation (Fig 1) Different isotopic compositions of precipi-tation events can further add to a profile of d2H and d18

O in the soil (Midwood et al 1998) Since frac-tionation does not normally occur during uptake of water, the d-value of xylem water may contain information about the depth of water uptake or source of water (e.g., ground or stream water) Once in the leaf, the water is isotopically enriched as a result of transpiration The ultimate d2H and d18

O of leaf water is influenced by stomatal and boundary layer conductances, vapor pres-sure difference, transpiration rate and the d-values of water vapor around the leaf

The H in photoassimilates stems from water and carries its isotopic composition during assimilation That is also the case with O, although in an indirect manner Assimilated O is derived from CO2, but its O is exchanged with H2O in the reaction CO2$ HCO3—catalyzed by carbonic anhydrase Assimilates thus carry the isotopic signal of leaf water During CO2 assim-ilation, substantial fractionation occurs for 2H (—117%), whereas fractionation during further metabolism works in the opposite direction (+158%) There is also isotopic enrichment of 18

O during assimilation and metabolism (+27%) However, the environmental effect on these fractionation processes is limited During synthesis of macromolecules from assimilates exchange of H and O with water occurs (Fig 1) This applies only for part of the atoms The

continued

perennials, and succulent perennials depend on summer rains (Fig 5) Plants that have an isotopic composition of their xylem water that is representa-tive of deep water are less water stressed and have higher transpiration rates and lower water-use effi-ciency(Sect 6) than species with a shallow-water isotopic signature

3.4 Roots Sense Moisture Gradients and Grow Toward Moist Patches

As with so many other fascinating phenomena in plants, Darwin (1880) already noticed that roots have the amazing ability to grow away from dry sites and toward wetter pockets in the soil: They

Box 3.3Continued

FIGURE1 Isotopic fractionation and exchange processes of H and O in a tree and its environment

fraction of exchange during cellulose synthesis in tree rings was estimated at 0.36 for H and 0.42 for O (Roden et al 2000) Intramolecular posi-tions have different degrees of exchange (Stern-berg et al 2006) which can be used for specific purposes

are hydrotropic Positive hydrotropism occurs due to inhibition of root cell elongation at the humid side of the root The elongation at the dry side is either unaffected or slightly stimulated, resulting in a cur-vature of the root and growth toward a moist patch (Takahashi 1994, Tsuda et al 2003) The root cap is most likely the site of hydrosensing (Takahashi & Scott 1993), but the exact mechanism of hydrotrop-ismis not known It involves an increase in cell-wall extensibility of the root cells that face the dry side (Sect 2.2 of Chapter on growth and allocation; Hirasawa et al 1997) The hydrotropic response is

stronger in roots of Zea mays (corn), which is a species that tolerates relatively dry soils, than it is in those of Pisum sativum(pea), and it shows a strong interaction with the root’s gravitropic response (Fig 6)

4 Water Relations of Cells

There are major constraints that limit the mechan-isms by which plants can adjust cellular water potential Adjustment of the water potential must come through variation in hydrostatic pressure or

FIGURE Hydrogen isotope ratios (dD) of xylem

water during the summer from plants of different growth forms in a Utah desert scrub community The mean winter precipitation dD was –88‰, whereas summer precipitation dD ranged from –22 to –80‰ (Ehleringer et al 1991)

FIGURE6 Hydrotropism in roots of Zea mays (corn) and

of the wild type and the ageotropic mutant (ageotro-pum) of Pisum sativum (pea) (A) Diagram showing the humidity-controlled chamber Roots were placed 2–3 mm from the ‘‘hydrostimulant’’ (wet cheesecloth) Saturated solutions of salts create the humidity gradient Different salts (KCl, K2CO3) give different gradients The

relative humidity and temperature was measured with a

thermohygrometer (P) A stationary hygrometer (S) measured the relative humidity in the chamber The arrow and letter g indicate the direction of gravitational force (B) Moisture gradients, between and 50 mm from the hydrostimulant, created by using no salt (a), KCl (b), or K2CO3(c) (C) Root curvature 10 hours after

the beginning of hydrostimulation by the three moisture gradients shown in (B) (after Takahashi & Scott 1993)